Mycobacterial proteins as early antigens for serodiagnosis and vaccines

ABSTRACT

In view of the paucity of human material available to study the immunological events occurring after inhalation of virulent bacilli, but prior to development of clinical TB, the present invention is based in part on studies of aerosol infected rabbits. The present inventors reasoned that by 3-5 weeks post-infection, the sera from infected rabbits would contain antibodies to the antigens being expressed by the in vivo bacteria.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the fields of microbiology and medicine relates tomethods for rapid early detection of mycobacterial disease in humansbased on the presence of antibodies to particular “early” mycobacterialantigens which have not been previously recognized for this purpose.Assay of such antibodies on select partially purified or purifiedmycobacterial preparations containing such early antigens permitsdiagnosis of TB earlier than has been heretofore possible. Also providedis a surrogate marker for screening populations at risk for TB, inparticular subjects infected with human immunodeficiency virus (HIV).The invention is also directed to vaccine compositions and methodsuseful for preventing or treating TB.

2. Description of the Background Art

The incidence of tuberculosis has shown a rapid increase in recentyears, not only in the developing countries, but also in crowded urbansettings in the US and in specific subsets of our society, including thehomeless, IV drug users, HIV-infected individuals, immigrants andrefugees from high prevalence endemic countries (Raviglione, M C et al.,1995. JAMA. 273:220-226). Studies show that these populations are at asignificantly greater risk of developing tuberculosis, and also serve asthe reservoir of infection for the community as a whole Raviglione, M Cet al., 1992, Bull World Health Organization. 70:515-526; Raviglione, MC et al., 1995. JAMA. 273:220-226). None of the currently used methodsfor diagnosis of tuberculosis identify individuals with active butsub-clinical infection, and the disease is generally detected when theindividuals are already infectious. Design of new diagnostic assaysrequires knowledge of antigens expressed by the bacteria during their invivo survival. Most current studies of antigens of Mycobacteriumtuberculosis (Mtb); also abbreviated herein are focused on antigenspresent in the culture filtrates of bacteria replicating actively invitro, with the presumption that the same molecules are expressed by thein vivo bacteria.

A vast majority of the Mtb infected individuals develop immune responsesthat arrest progression of infection to clinical TB, and also preventthe latent bacilli from reactivating to cause clinical disease, whereasabout 10-15% of the infected individuals progress to developing primaryor reactivation TB. Understanding the host-pathogen interactions thatoccur after infection, but prior to development of clinical TB(pre-clinical TB) is required both for the design of effective vaccinesand for development of diagnosis of early disease.

Several studies have shown that Mtb adapts to different environments inbroth media

-   (Garbe, T R et al., 1999, Infect. Immun. 67:460-465; Lee, B-Y et    al., 1995, J. Clin. Invest. 96:245-249; Wong, D K et al., 1999,    Infect. Immun. 67:327-336) and during intracellular residence by    altering its gene expression (8, 22, 34).-   Clark-Curtiss, J E et al., 1999, p. 206-210. In Proceedings of    Thirty-Fourth Tuberculosis-Leprosy Research Conference, San    Francisco, Calif., Jun. 27-30.-   Lee et al., supra; Smith, I et al., 1998, Tuber. Lung Dis.    79:91-97). Earlier studies from the present inventors' laboratory    with cavitary and non-cavitary TB patients have also shown that the    in vivo environment in which the bacilli replicate affects the    profile of the antigenic proteins expressed by Mtb (Samanich, K M et    al., 1998, J. Infect. Dis. 178:1534-1538; Laal et al., U.S. Pat. No.    6,245,331 (2001)).

One objective of the present invention was to identify the antigensexpressed by inhaled Mtb during the pre-clinical stages of TB. There areno markers to identify non-diseased humans with an active infection withMtb, but the rabbit model of TB closely resembles TB in immuno-competenthumans in that both species are outbred, both are relatively resistantto Mtb, and in both the caseous lesions may liquify and form cavities(Converse, P J et al., 1996, Infect. Immun. 64:4776-4787). Studies haveshown that on being inhaled, the bacilli are phagocytosed by (nonspecifically) activated alveolar macrophages (AM) which either destroyor allow them to multiply. If the bacilli multiply, the AM die and thereleased bacilli are phagocytosed by non activated monocyte/macrophagesthat emigrate from the bloodstream. Intracellular replication and hostcell death continue for 3-5 weeks, when both cellular and humoral immuneresponses are elicited (Lurie, M B, 1964. Chapter VIII, p. 192-222, InM. B. Lurie (ed.) Resistance to tuberculosis: experimental studies innative and acquired defensive mechanisms. Harvard University Press,Cambridge, Mass.; Lurie, M B et al., 1965, Bact. Rev. 29:466-476;Dannenberg, A M., Jr., 1991, Immunol. Today. 12:228-233). Lymphocytesand macrophages enter the foci of infection, and if they becomeactivated bacillary replication is controlled, if not, the infectionprogresses to clinical disease. During these initial stages of bacillaryreplication and immune stimulation, there are no outward signs ofdisease except the conversion of cutaneous reactivity to PPD. Theantigens of Mtb expressed, and their interaction with the immune systemduring these pre-clinical stages of TB is not delineated.

SUMMARY OF THE INVENTION

In view of the paucity of human material available to study theimmunological events occurring after inhalation of virulent bacilli, butprior to development of clinical TB, the present invention is based inpart on studies of aerosol infected rabbits. The present inventorsreasoned that by 3-5 weeks post-infection, the sera from infectedrabbits would contain antibodies to the antigens being expressed by thein vivo bacteria.

Four antigens of Mtb that are expressed in vivo after aerosol infection,but prior to development of clinical TB, in rabbits were identified byimmunoscreening an expression library of Mtb genomic DNA with seraobtained 5 weeks post-infection. Three of the proteins identified, PirG(Rv3810) [SEQ ID NO:1 and 2; nucleotide and amino acid], PE-PGRS(Rv3367) [SEQ ID NO:3 and 4] and PTRP (Rv0538) [SEQ ID NO:5 and 6] havemultiple tandem repeats of unique amino-acid sequences, and havecharacteristics of surface or secreted proteins. The fourth protein,MtrA (Rv3246c) [SEQ ID NO:7 and 8], is a response regulator of aputative two-component signal transduction system, mtrA-mtrB, of Mtb.All four antigens were recognized by pooled sera from TB patients andnot from healthy controls, confirming their in vivo expression duringactive infection in humans. Three of the antigens, (PE-PGRS, PTRP andMtrA) were also recognized by retrospective, pre-clinical TB seraobtained from HIV-TB patients prior to the clinical manifestation of TB,suggesting their utility as diagnostics for active, pre-clinical(“early”) TB.

The present invention provides methods, kits and compositions directedto the detection of antibodies or T cell reactivity to any of the aboveearly antigens or to the detection of the antigens themselves in a bodyfluid of a subject as a means of detecting early mycobacterial diseasein the subject. In other embodiments, the invention provides, methods,kits and compositions useful for detecting antibody or T cell reactivityto, in addition to one or more of the above early antigens, to one ormore of the following early Mtb antigens:

(a) an 88 kDa M. tuberculosis protein having the an amino acid sequenceSEQ ID NO:13: MTDRVSVGNL RIARVLYDFV NNEALPGTDI DPDSFWAGVD KVVADLTPQNQALLNARDEL QAQIDKWHRR RVIEPIDMDA YRQFLTEIGY LLPEPDDFTI TTSGVDAEITTTAGPQLVVP VLNARFALNA ANARWGSLYD ALYGTDVIPE TDGAEKGPTY NKVRGDKVIAYARKFLDDSV PLSSGSFGDA TGFTVQDGQL VVALPDKSTG LANPGQFAGY TGAAESPTSVLLINHGLHIE ILIDPESQVG TTDRAGVKDV ILESAITTIM DFEDSVAAVD AADKVLGYRNWLGLNKGDLA AAVDKDGTAF LRVLNRDRNY TAPGGGQFTL PGRSLMFVRN VGHLMTNDAIVDTDGSEVFE GIMDALFTGL IAIHGLKASD VNGPLINSRT GSIYIVKPKM HGPAEVAFTCELFSRVEDVL GLPQNTMKIG IMDEERRTTV NLKACIKAAA DRVVFINTGF LDRTGDEIHTSMEAGPMVRK GTMKSQPWIL AYEDHNVDAG LAAGFSGRAQ VGKGMWTMTE LMADMVETKIAQPRAGASTA WVPSPTAATL HALHYHQVDV AAVQQGLAGK RRATIEQLLT IPLAKELAWAPDEIREEVDN NCQSILGYVV RWVDQGVGCS KVPDIHDVAL MEDRATLRIS SQLLANWLRHGVITSADVRA SLERMAPLVD RQNAGDVAYR PMAPNFDDSI AFLAAQELIL SGAQQPNGYTEPILHRRRRE FKARAAEKPA PSDRAGDDAA R

(b) a 27 kDa M. tuberculosis protein named MPT51 having the amino acidsequence SEQ ID NO:14: APYENLMVPS PSMGRDIPVA FLAGGPHAVY LLDAFNAGPDVSNWVTAGNA NTLAGKGIS VVAPAGGAYS MYTNWEQDGS KQWDTFLSAE LPDWLAANRGAAQGGYGAMA AAFHPDRFG FAGSMSGFLY PSNTTTNGAI AAGMQQFGGV DTNGMWGAPQLGRWKWHDPW HASLLAQNN TRVWVWSPTN PGASDPAAMI GQTAEAMGNS RMFYNQYRSVGGHNGHFDFP SGDNGWGSW APQLGAMSGD IVGAIR;

-   (c) a protein characterized as M. tuberculosis antigen 85C; or-   (d) a glycoprotein characterized as M. tuberculosis antigen MPT32.

In yet another embodiment, the invention provides methods, kits andcompositions useful for the detection of antibodies or T cell reactivityto any of the above early antigens or to one or more of the followingearly antigens:

(i) a 28 kDa protein corresponding to the spot identified as Ref. No. 77in Table 2.

(ii) a 29/30 kDa protein corresponding to the spot identified as Ref No.69 or 59 in Table 2;

(iii) a 31 kDa protein corresponding to the spot identified as Ref. No.103 in Table 2;

(iv) a 35 kDa protein corresponding to the spot identified as Ref. No.66 in Table 2 and reacting with monoclonal antibody IT-23;

(v) a 42 kDa protein corresponding to the spot identified as Ref. No. 68or 80 in Table 2;

(vi) a 48 kDa protein corresponding to the spot identified as Ref. No.24 in Table 2; and

(vii) a 104 kDa protein corresponding to the spot identified as Ref. No.111 in Table 2, which spots are obtained by 2-dimensionalelectrophoretic separation of M. tuberculosis lipoarabinomannan-freeculture filtrate proteins as follows:

-   -   (A) incubating 3 hours at 20° C. in 9M urea, 2% Nonidet P-40, 5%        β-mercaptoethanol, and 5% ampholytes at pH 3-10;    -   (B) isoelectric focusing on 6% polyacrylamide isoelectric        focusing tube gel of 1.5 mm×6.5 cm, said gel containing 5%        ampholytes in a 1:4 ratio of pH 3-10 ampholytes to pH 4-6.5        ampholytes for 3 hours at 1 kV using 10 mM H₃PO₄ as catholyte        and 20 mM NaOH as anolyte, to obtain a focused gel;    -   (C) subjecting the focused gel to SDS PAGE in the second        dimension by placement on a preparative SDS-polyacrylamide gel        of 7.5×10 cm×1.5 mm containing a 6% stack over a 15% resolving        gel and electrophoresing at 20 mA per gel for 0.3 hours followed        by 30 mA per gel for 1.8 hours.

In yet other embodiments, the present invention provides vaccinescompositions and methods for treating or preventing mycobacterialdisease in a subject. The vaccine composition may comprise any one ormore of the early antigens noted above or an epitope thereof. Preferredvaccine epitopes are T helper epitopes, more preferably T helperepitopes that stimualte Th1 cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows reactilvit8y of guinea pig serum pool with antigen for Mtb(see description below figure)

FIGS. 2 a-2 d show reactivity of fusion proteins from individualcolonies of gsrI-3, I-6, II-1 and II-1 (see description below figure)

FIG. 3 a shows Western blots of fusion proteins with antibodies toβ-galactosidase (see description below figure)

FIG. 3 b shows reactivity of sera from Mtb-infected guinea pigs andant-β-gal antibody with fractionated lysates (see description belowfigure).

FIG. 4 a shows sequence alignment of clones gsr II-2 and I-6 with cosmidMTV004.

FIG. 4 b shows the amino acid sequence of protein encoded byMTV-=004.03. Peptides encoded by clones gsr I-6 and gsr II-2 are shownin bold. The 6 copies of the repeat motif in gsr I-6 are underlined.

FIG. 5 a shows sequence alignment of clone gsr II-1 with cosmid MTCY336.

FIG. 5 b shows amino acid sequence of MTCY336.28. Peptide encoded byclone gsr II-2 is shown in bold.

FIGS. 6A, 6B and 6C shows reactivit8y of fusion proteins of gsrI-6, II-1and II-1 with sera from individual guinea pigs (see description belowfigures).

FIGS. 7A and 7B show a comparison of reactivity of Mtb infected guineapig and human sera with culture filtrate proteins (7A) and SDS-solublecell wall proteins (7B) of Mtb (see description below figure).

FIGS. 8A and 8B shows reactivity of a pool of sera from PPD+ healthyindividuals (8A) or from TB patients (8B) (see description below figure)

FIG. 9 shows reactivity of sera from PPD+ individuals and TB patientswith gsr I-6 lysates (see description below figure).

FIG. 10 ( shows reactivity of HIV pre-TB serum pool with fusion proteinsexpressed by the various gsr clones (see description below figure).

FIG. 11 shows a comparison of reactivity of TB sera with native andrecombinant Mtb antigens (see description below figure).

FIG. 12A shows expression of the 88 kDa seroreactive antigen in M.smegmatis (see description below figure).

FIG. 12B shows reactivity of sera form a TB patient and a PPD+ healthycontrol with 30-fold concentrated culture filtrate of M. smegmatis (seedescription below figure).

FIG. 13 Reactivity of Mtb antigens with pooled sera from rabbits. LFCFP(lanes 2 & 3 and SDS-CWP (lanes 4 & 5) proteins of Mtb were fractionatedon 10% SDS-PA gels, and western blots probed with pooled sera fromuninfected (lanes 2 & 4) and Mtb infected (lanes 3 & 5) rabbits. Lane 1contains molecular weight markers.

FIG. 14: Reactivity of β-gal fusion proteins of AD clones with antiβ-gal antibody and sera from Mtb infected rabbits. Lysates of ADlysogens and λgt11 vector lysogen were separated on 10% SDS-PA gels andprobed with anti-β-gal antibody (lanes 2-9), uninfected rabbit sera(lanes 11-18) and infected rabbit sera (lanes 19-27). Lanes; 1, 10 & 19contain molecular weight markers; lanes 2, 11 & 20: lysates from cloneAD 1; lanes 3, 12 & 21: clone AD2, lanes 4; 13 &22: clone AD4; lanes 5,14 & 23: clone AD9; lanes 6, 15 & 24: clone AD10; lanes 7, 16 & 25:clone AD7; lanes 8, 17 & 26: clone AD16 and lanes 9, 18 & 27: λgt11vector.

FIG. 15: Schematic maps showing position of AD clones on cosmids of MtbH37Rv. A: map of clones AD1 & AD2 on cosmid MTV026 and MTCY409. B: cloneAD9 on cosmid MTV004. C: clone AD10 on cosmid MTY25D10. D: clone AD16 oncosmid MTY20B11. Black bar represents the gene on the cosmid. Hatchedbar shows regions expressed as β-gal fusion protein in AD clones. Arrowindicates direction of translation. E denotes EcoRI site.

FIG. 16: Nucleotide and deduced amino acid sequence of gene Rv3367(PE_PGRS) (SEQ ID NO: 3 and 4, respectively). The signal peptidesequence is shown in italics, hollow arrow between aa 44 & 45 indicatessignal peptidase cleavage site. The repetitive sequences are shown inboxes. The motif PE is underlined. Solid arrow at aa 230 indicates thestart of fusion with β-gal in clone AD9. The transmembrane helicessequences are shown in bold. The asterisk indicates the terminationcodon.

FIG. 17: Nucleotide and deduced amino acid sequence of gene Rv0538(PTRP) (SEQ ID NO: 5 and 6, respectively). The repetitive motifs areshown in boxes. Arrow indicates the initiation of fusion with β-gal inclone AD10. The transmembrane helices sequences are shown in bold. Theasterisk indicates the termination codon.

FIG. 18: Reactivity of β-gal fusion proteins with human sera. Blot A:clone AD9 (PE_PGRS), B: clone AD10 (PTRP), C: clone AD2 (pirG) and D:clone AD16 (MtrA). Lanes 1, 4, 7, 10 & 13: molecular weight markers;lanes 2, 5, 8, 11 & 14: lysates form lysogens of respective AD clones;lanes 3, 6, 9, 12 & 15: lysogen of λgt11 vector. Lanes 2 & 3 probed withanti β-gal antibody, lanes 5 & 6 with pooled sera from PPD positivehealthy individuals, lanes 8 & 9 with pooled sera from HIV pre-TBindividuals, lanes 11 & 12 with pooled sera from non-cavitary TBindividuals and lanes 14 & 15 with pooled sera from cavitary TBindividuals.

FIG. 19: Reactivity of β-gal fusion proteins of AD clones with sera fromHIV pre-TB individuals. A: clone AD9 (PE_PGRS), B: clone AD10 (PTRP) andC: clone AD16 (MtrA). Lane 1; molecular weight marker; lanes 2-15:lysates from lysogens of respective AD clones. Lane 2 is probed withanti β-gal antibody in each case, lanes 3-5 with sera from three PPDpositive healthy individuals and lanes 6-15 with sera from 10 HIV pre-TBindividuals.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This Application incorporates by reference, in their entirety, U.S. Pat.No. 6,245,331 (12 Jun. 2001) and Laal et al., U.S. Ser. No. 9/396,347(filed Sep. 14, 1999) 09/001,984, filed 31 Dec. 1997, which claimspriority from U.S. Ser. No. 60/034,003, filed 31 Dec. 31, 1996). Alsoincorporated by reference are all references cited therein.

In the following description, reference will be made to variousmethodologies known to those of skill in the art of immunology.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reference works settingforth the general principles of immunology include Roitt, I., EssentialImmunology, 6th Ed., Blackwell Scientific Publications, Oxford (1988);Roitt, I. et al., Immunology, C. V. Mosby Co., St. Louis, Mo. (1985);Klein, J., Immunology, Blackwell Scientific Publications, Inc.,Cambridge, Mass., (1990); Klein, J., Immunology: The Science ofSelf-Nonself Discrimination, John Wiley & Sons, New York, N.Y. (1982));and Eisen, H. N., (In: Microbiology, 3rd Ed. (Davis, B. D., et al.,Harper & Row, Philadelphia (1980)); A standard work setting forthdetails of mAb production and characterization, and immunoassayprocedures, is Hartlow, E. et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988.

As used herein, the term “early” and “late” in reference to (1) Mtbinfection or disease, or the subject having the infection or disease,(2) the antibody response to an Mtb antigen, (3) an Mtb antigen itselfor (4) a diagnostic assay, are defined in terms of the stage ofdevelopment of TB. Early and late (or advanced) TB are defined in thetable below.

Thus, a subject with early TB is asymptomatic or, more typically, hasone or more “constitutional symptoms” (e.g., fever, cough, weight loss).In early TB, Mtb bacilli are too few to be detectable as acid-fastbacilli in smears of sputum or other body fluid, primarily those fluidsassociated with the lungs (such as bronchial washings, bronchoalveolarlavage, pleural effusion). However, in these subjects, Mtb bacilli arepresent and culturable, i.e., can be grown in culture from the abovebody fluids. Finally, early TB subjects may have radiographicallyevident pulmonary lesions which may include infiltration but withoutcavitation. Any antibody present in such early stages is termed an“early antibody” and any Mtb antigen recognized by such antibodies istermed an “early antigen.” The fact that an antibody is characterized as“early” does not mean that this antibody is absent in advanced TB.Rather, such antibodies are expected to persist across the progressionof early TB to the advanced stage. Early 1. Smear of sputum, bronchialwashing, bronchoalveolar TB    lavage or pleural effusion is negativefor acid fast bacilli 2. Direct culture of sputum, bronchial washing,   bronchoalveolar lavage or pleural effusion is    positive for acid fastbacilli 3. Chest x-ray is normal or shows Infiltration in the lungs 4.Constitutional symptoms are present (fever, cough, appetite    andweight loss) Late/ 1. Smear of sputum, bronchial washing,bronchoalveolar Advanced    lavage or pleural effusion is positive TB   (with possible hemoptysis) 2. Direct culture of sputum, bronchialwashing,    bronchoalveolar lavage or pleural effusion is positive 3.Chest x-ray shows cavitary lesions in the lungs 4. Constitutionalsymptoms are present (see above)

Accordingly, the term “late” or “advanced” is characterized in that thesubject has frank clinical disease and more advanced cavitary lesions inthe lungs. In late TB, Mtb bacilli are not only culturable from smearsof sputum and/or the other body fluids noted above, but also present insufficient numbers to be detectable as acid-fast bacilli in smears ofthese fluids. Again, “late TB” or “late mycobacterial disease” is usedinterchangeably with “advanced TB” or “advanced mycobacterial disease.”An antibody that first appears after the onset of diagnostic clinicaland other characterizing symptoms (including cavity pulmonary lesions)is a late antibody, and an antigen recognized by a late antibody (butnot by an early antibody) is a late antigen.

To be useful in accordance with this invention, an early diagnosticassay must permit rapid diagnosis of Mtb disease at a stage earlier thanthat which could have been diagnosed by conventional clinical diagnosticmethods, namely, by radiologic examination and bacterial smear andculture or by other laboratory methods available prior to thisinvention. (Culture positivity is the final confirmatory test but takestwo weeks and more)

An objective of the invention is to define, obtain and characterize theantigens of Mtb expressed by the bacterium in vivo during earlytuberculosis. These antigens are evaluated for their utility as markersof early disease that may be used to monitor suspected or high-riskindividuals to identify those with active, subclinical infection.

Mycobacterial Antigen Compositions

The preferred mycobacterial antigen composition may be a substantiallypurified or recombinantly produced preparation of one or more Mtbproteins. Alternatively, the antigen composition may be a partiallypurified or substantially pure preparation containing one or more Mtbepitopes which are capable of being bound by antibodies or T lymphocytesof an infected subject

Such epitopes may be in the form of peptide fragments of the earlyantigen proteins or other “functional derivatives” of Mtb proteins asdescribed below.

By “functional derivative” is meant a “fragment,” “variant,” “analogue,”or “chemical derivative” of an early antigen protein, which terms aredefined below. A functional derivative retains at least a portion of thefunction of the protein which permits its utility in accordance with thepresent invention—primarily the capacity to bind to an early antibody. A“fragment” refers to any subset of the molecule, that is, a shorterpeptide. A “variant” refers to a molecule substantially similar toeither the entire protein or fragment thereof A variant peptide may beconveniently prepared by direct chemical synthesis or by recombinantmeans. An “analogue” of the protein or peptide refers to a non- naturalmolecule substantially similar to either the entire molecule or afragment thereof. A “chemical derivative” of the antigenic protein orpeptide contains additional chemical moieties not normally part of thepeptide. Covalent modifications of the peptide are included within thescope of this invention. Such modifications may be introduced into themolecule by reacting targeted amino acid residues of the peptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or terminal residues.

Several proteins or glycoproteins, identified in culture filtrates ofMt, or on the surface of Mtb organisms are the preferred early Mtbantigens of the present invention. The secreted proteins may also bepresent in cellular preparations of the bacilli. Thus, these earlyantigens are not intended to be limited to the secreted protein form.The proteins are characterized at various places below.

Preferred diagnostic epitopes are those recognized by antibodies or by Tcells, preferably Th1 cells of “early” TB patients as defined above.This does not exclude the possibility that such epitopes are bound byantibodies or recognized by T cells present later in the infectiousprocess. In fact, some of the present proteins or epitopes thereof mydetect infection in subjects whose infectious state is not detected byantibodies against the 88 kDa protein (malate synthase) described inU.S. Pat. No. 6,245,331 and U.S. Ser. No. 9/396,347, and theirrespective file histories.

Preferred vaccine epitopes (see below) are epitopes which stimulatenaïve human Th1 cells or Th1 cells or infected subjects to proliferateor to secrete cytokines. Assays for Th1 cytokines, preferablyinterferon-γ (IFNγ). IL-12 and IL-18 are well-known in the art.

The present immunoassay typically comprises incubating a biologicalfluid, preferably serum or urine, from a subject suspected of having TB,in the presence of an Mtb antigen-containing reagent which includes oneor more Mtb early antigens, and detecting the binding of antibodies inthe sample to the mycobacterial antigen(s). By the term “biologicalfluid” is intended any fluid derived from the body of a normal ordiseased subject which may contain antibodies, such as blood, serum,plasma, lymph, urine, saliva, sputum, tears, cerebrospinal fluid,bronchioalveolar lavage fluid, pleural fluid, bile, ascites fluid, pusand the like. Also included within the meaning of this term as usedherein is a tissue extract, or the culture fluid in which cells ortissue from the subject have been incubated.

In a preferred embodiment, the mycobacterial antigen composition isbrought in contact with, and allowed to bind to, a solid support orcarrier, such as nitrocellulose or polystyrene, allowing the antigencomposition to adsorb and become immobilized to the solid support. Thisimmobilized antigen is then allowed to interact with the biologicalfluid sample which is being tested for the presence of anti-Mtbantibodies, such that any antibodies in the sample will bind to theimmobilized antigen. The support to which the antibody is now bound maythen be washed with suitable buffers after which a detectably labeledbinding partner for the antibody is introduced. The binding partnerbinds to the immobilized antibody. Detection of the label is a measureof the immobilized antibody.

A preferred binding partner for this assay is an anti-immunoglobulinantibody (“second antibody”) produced in a different species. Thus todetect a human antibody, a detectably labeled goat anti-humanimmunoglobulin “second” antibody may be used. The solid phase supportmay then be washed with the buffer a second time to remove unboundantibody. The amount of bound label on the solid support may then bedetected by conventional means appropriate to the type of label used(see below).

Such a “second antibody” may be specific for epitopes characteristic ofa particular human immunoglobulin isotype, for example IgM, IgG₁,IgG_(2a), IgA and the like, thus permitting identification of theisotype or isotypes of antibodies in the sample which are specific forthe mycobacterial antigen. Alternatively, the second antibody may bespecific for an idiotype of the ant-Mtb antibody of the sample.

As alternative binding partners for detection of the sample antibody,other known binding partners for human immunoglobulins may be used.Examples are the staphylococcal immunoglobulin binding proteins, thebest know of which is protein A. Also intended is staphylococcal proteinG, or a recombinant fusion protein between protein A and protein G.Protein G of group G and group C streptococci binds to the Fc portion ofIg molecules as well as to IgG Fab fragment at the V_(H)3 domain.Protein C of Peptococcus magnus binds to the Fab region of theimmunoglobulin molecule. Any other microbial immunoglobulin bindingproteins, for example from Streptococci, are also intended (for example,Langone, J. J., Adv. Immunol. 32:157 (1982)).

In another embodiment of this invention, a biological fluid suspected ofcontaining antibodies specific for a Mtb antigen may be brought intocontact with a solid support or carrier which is capable of immobilizingsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with a mycobacterial antigen reagent, which may bedetectably labeled. Bound antigen is then measured by measuring theimmobilized detectable label. If the mycobacterial antigen reagent isnot directly detectably labeled, a second reagent comprising adetectably labeled binding partner for the Mtb antigen, generally asecond anti-Mtb antibody such as a murine mAb, is allowed to bind to anyimmobilized antigen. The solid phase support may then be washed withbuffer a second time to remove unbound antibody. The amount of boundlabel on said solid support may then be detected by conventional means.

By “solid phase support” is intended any support capable of binding aproteinaceous antigen or antibody molecules or other binding partnersaccording to the present invention. Well-known supports, or carriers,include glass, polystyrene, polypropylene, polyethylene, polyvinylidenedifluoride, dextran, nylon, magnetic beads, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor the purposes of the present invention. The support material may havevirtually any possible structural configuration so long as it is capableof binding to an antigen or antibody. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads, 96-well polystyrene microplates and teststrips, all well-known in the art. Those skilled in the art will knowmany other suitable carriers for binding antibody or antigen, or will beable to ascertain the same by use of routine experimentation.

Using any of the assays described herein, those skilled in the art willbe able to determine operative and optimal assay conditions for eachdetermination by employing routine experimentation. Furthermore, othersteps as washing, stirring, shaking, filtering and the like may be addedto the assays as is customary or necessary for the particular situation.

A preferred type of immunoassay to detect an antibody specific for amycobacterial antigen according to the present invention is anenzyme-linked immunosorbent assay (ELISA) or more generically termed anenzyme immunoassay (EIA). In such assays, a detectable label bound toeither an antibody-binding or antigen-binding reagent is an enzyme. Whenexposed to its substrate, this enzyme will react in such a manner as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Enzymes which can beused to detectably label the reagents useful in the present inventioninclude, but are not limited to, horseradish peroxidase, alkalinephosphatase, glucose oxidase, β-galactosidase, ribonuclease, urease,catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase,Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphatedehydrogenase, triose phosphate isomerase, glucose-6-phosphatedehydrogenase, glucoamylase and acetylcholinesterase. For descriptionsof EIA procedures, see Voller, A. et al., J. Clin. Pathol. 31:507-520(1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E.(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980; Butler, J. E.,In: Structure of Antigens, Vol. 1 (Van Regenmortel, M., CRC Press, BocaRaton, 1992, pp. 209-259; Butler, J. E., In: van Oss, C. J. et al.,(eds), Immuniochemistry, Marcel Dekker, Inc., New York, 1994, pp.759-803; Butler, J. E. (ed.), Immunochemistry of Solid-PhaseImmunoassay, CRC Press, Boca Raton, 1991)

In another embodiment, the detectable label may be a radiolabel, and theassay termed a radioimmunoassay (RIA), as is well known in the art. See,for example, Yalow, R. et al., Nature 184:1648 (1959); Work, T. S., etal., Laboratory Techniques and Biochemistry in Molecular Biology, NorthHolland Publishing Company, NY, 1978, incorporated by reference herein.The radioisotope can be detected by a gamma counter, a scintillationcounter or by autoradiography. Isotopes which are particularly usefulfor the purpose of the present invention are ¹²⁵I, ¹³¹I, ³⁵S, ³H and¹⁴C.

It is also possible to label the antigen or antibody reagents with afluorophore. When the fluorescently labeled antibody is exposed to lightof the proper wave length, its presence can then be detected due tofluorescence of the fluorophore. Among the most commonly usedfluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine orfluorescence-emitting metals such as ¹⁵²Eu or other lanthanides. Thesemetals are attached to antibodies using metal chelators.

The antigen or antibody reagents useful in the present invention alsocan be detectably labeled by coupling to a chemiluminescent compound.The presence of a chemiluminescent-tagged antibody or antigen is thendetermined by detecting the luminescence that arises during the courseof a chemical reaction. Examples of useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescentcompound such as a bioluminescent protein may be used to label theantigen or antibody reagent useful in the present invention. Binding ismeasured by detecting the luminescence. Useful bioluminescent compoundsinclude luciferin, luciferase and aequorin.

Detection of the detectably labeled reagent according to the presentinvention may be accomplished by a scintillation counter, for example,if the detectable label is a radioactive gamma emitter, or by afluorometer, for example, if the label is a fluorophore. In the case ofan enzyme label, the detection is accomplished by colorimetry to measurethe colored product produced by conversion of a chromogenic substrate bythe enzyme. Detection may also be accomplished by visual comparison ofthe colored product of the enzymatic reaction in comparison withappropriate standards or controls.

The immunoassay of this invention may be a “two-site” or “sandwich”assay. The fluid containing the antibody being assayed is allowed tocontact a solid support. After addition of the mycobacterial antigen(s),a quantity of detectably labeled soluble antibody is added to permitdetection and/or quantitation of the ternary complex formed betweensolid-phase antibody, antigen, and labeled antibody. Sandwich assays aredescribed by Wide, Radioimmune Assay Method, Kirkham et aL, Eds., E. &S. Livingstone, Edinburgh, 1970, pp 199-206.

Alternatives to the RIA and EIA are various types of agglutinationassays, both direct and indirect, which are well known in the art. Inthese assays, the agglutination of particles containing the antigen(either naturally or by chemical coupling) indicates the presence orabsence of the corresponding antibody. Any of a variety of particles,including latex, charcoal, kaolinite, or bentonite, as well as microbialcells or red blood cells, may be used as agglutinable carriers (Mochida,U.S. Pat. No. 4,308,026; Gupta et al., J. Immunol. Meth. 80:177-187(1985); Castelan et al., J. Clin. Pathol. 21:638 (1968); Singer et al.,Amer. J. Med.(December 1956, 888; Molinaro, U.S. Pat. No. 4,130,634).Traditional particle agglutination or hemagglutination assays aregenerally faster, but much less sensitive than RIA or EIA. However,agglutination assays have advantages under field conditions and in lessdeveloped countries.

In addition to detection of antibodies, the present invention providesmethods to detect and enumerate cells secreting an antibody specific fora mycobacterial antigen. Thus, for example, any of a number of plaque orspot assays may be used wherein a sample containing lymphocytes, such asperipheral blood lymphocytes, is mixed with a reagent containing theantigen of interest. As the antibody secreting cells of the samplesecrete their antibodies, the antibodies react with the antigen, and thereaction is visualized in such a way that the number of antibodysecreting cells (or plaque forming cells) may be determined. The antigenmay be coupled to indicator particles, such as erythrocytes, preferablysheep erytirocytes, arranged in a layer. As antibodies are secreted froma single cell, they attach to the surrounding antigen-bearingerythrocytes. By adding complement components, lysis of the erythrocytesto which the antibodies have attached is achieved, resulting in a “hole”or “plaque” in the erythrocyte layer. Each plaque corresponds to asingle antibody-secreting cell. In a different embodiment, the samplecontaining antibody-secreting cells is added to a surface coated with anantigen-bearing reagent, for example, a mycobacterial antigen alone orconjugated to bovine serum albumin, attached to polystyrene. After thecells are allowed to secrete the antibody which binds to the immobilizedantigen, the cells are gently washed away. The presence of a colored“spot” of bound antibody, surrounding the site where the cell had been,can be revealed using modified EIA or other staining methods well-knownin the art. (See, for example, Sedgwick, J. D. et aL, J. Immunol. Meth.57:301-309 (1983); Czerkinsky, C. C. et al., J. Immunol. Meth.65:109-121 (1983); Logtenberg, T. et al., Immunol. Lett. 9:343-347(1985); Walker, A. G. et al., J. Immunol. Meth. 104:281-283 (1987).

The present invention is also directed to a kit or reagent system usefulfor practicing the methods described herein. Such a kit will contain areagent combination comprising the essential elements required toconduct an assay according to the disclosed methods. The reagent systemis presented in a commercially packaged form, as a composition oradmixture (where the compatibility of the reagents allow), in a testdevice configuration, or more typically as a test kit. A test kit is apackaged combination of one or more containers, devices, or the likeholding the necessary reagents, and usually including writteninstructions for the performance of assays. The kit may includecontainers to hold the materials during storage, use or both. The kit ofthe present invention may include any configurations and compositionsfor performing the various assay formats described herein.

For example, a kit for determining the presence of anti-Mtb earlyantibodies may contain one or more early Mtb antigens, either inimmobilizable form or already immobilized to a solid support, and adetectably labeled binding partner capable of recognizing the sampleanti-Mtb early antibody to be detected, for example. a labeledanti-human Ig or anti-human Fab antibody. A kit for determining thepresence of an early Mtb antigen may contain an immobilizable orimmobilized “capture” antibody which reacts with one epitope of an earlyMtb antigen, and a detectably labeled second (“detection”) antibodywhich reacts with a different epitope of the Mtb antigen than thatrecognized by the (capture) antibody. Any conventional tag or detectablelabel may be part of the kit, such as a radioisotope, an enzyme, achromophore or a fluorophore. The kit may also contain a reagent capableof precipitating immune complexes.

A kit according to the present invention can additionally includeancillary chemicals such as the buffers and components of the solutionin which binding of antigen and antibody takes place.

The present invention permits isolation of an Mtb early antigen which isthen used to produce one or more epitope-specific mAbs, preferably inmice. Screening of these putative early Mtb-specific mAbs is done usingknown patient sera which have been characterized for their reactivitywith the early antigen of interest. The murine mAbs produced in this wayare then employed in a highly sensitive epitope-specific competitionimmunoassay for early detection of TB. Thus, a patient sample is testedfor the presence of antibody specific for an early epitope of Mtb by itsability to compete with a known mAb for binding to a purified earlyantigen. For such an assay, the mycobacterial preparation may be lessthan pure because, under the competitive assay conditions, the mAbprovides the requisite specificity for detection of patient antibodiesto the epitope of choice (for which the mAb is specific).

In addition to the detection of early Mtb antigens or early antibodies,the present invention provides a method to detect immune complexescontaining early Mtb antigens in a subject using an EIA as describedabove. Circulating immune complexes have been suggested to be ofdiagnostic value in TB. (See, for example, Mehta, P. K. et al, 1989,Med. Microbiol. Immunol. 178:229-233; Radhakrishnan, V. V. et al., 1992,J. Med. Microbiol. 36:128-131). Methods for detection of immunecomplexes are well-known in the art. Complexes may be dissociated underacid conditions and the resultant antigens and antibodies detected byimmunoassay. See, for example, Bollinger, R. C. et al, 1992, J. Infec.Dis. 165:913-916. Immune complexes may be precipitated for directanalysis or for dissociation using known methods such as polyethyleneglycol precipitation.

Purified Mtb early antigens as described herein are preferably producedusing recombinant methods. See Examples. Conventional bacterialexpression systems utilize Gram negative bacteria such as E. coli orSalmonella species. However, it is believed that such systems are notideally suited for production of Mtb antigens (Burlein, J. E., In:Tuberculosis: Pathogenesis, Protection and Control, B. Bloom, ed., Amer.Soc. Microbiol., Washington, DC, 1994, pp. 239-252). Rather, it ispreferred to utilize homologous mycobacterial hosts for recombinantproduction of early Mtb antigenic proteins or glycoproteins. Methods forsuch manipulation and gene expression are provided in Burlein, supra.Expression in mycobacterial hosts, in particular M. bovis (strain BCG)or M. smegmatis are well-known in the art. Two examples, one ofmycobacterial genes (Rouse, D. A. et al., 1996, Mol. Microbiol.22:583-592) and the other of non mycobacterial genes, such as HIV-1genes (Winter, N. et al., 1992, Vaccines 92, Cold Spring Harbor Press,pp. 373-378) expressed in mycobacterial hosts are cited herein as anexample of the state of the art. The foregoing three references arehereby incorporated by reference in their entirety.

Urine-Based Antibody Assay

The present invention also provides a urine based diagnostic method forTB that can be used either as a stand-alone test, or as an adjunct tothe serodiagnostic methods described herein. Such a method enables thepractitioner to (1) determine the presence of anti-mycobacterialantibodies in urine from TB patients with early disease (non-cavitary,smear negative TB patients) and from HIV-infected TB patients; (2)determine the profile of specific mycobacterial antigens, such as thosein the culture filtrate, that are consistently and strongly reactivewith the urine antibodies; and (3) obtain the antigens that arerecognized by the urine antibodies.

Smear positive (=late) cases constitute only about 50% of the TB cases,and patients with relatively early disease are generally defined asbeing smear negative. Moreover, as the HIV-epidemic spreads indeveloping countries, the numbers and proportions of HIV-infected TBpatients increases.

Serum and urine samples from non-cavitary and/or smear negative, culturepositive TB patients and from HIV-infected TB patients are obtainedCohorts comprising PPD-positive and PPD-negative healthy individuals,non-tuberculous HIV-infected individuals, or close contacts of TBpatients can serve as negative controls.

The reactivity of the serum samples with culture filtrate proteins ofMtb, and the purified antigens (as described herein) is preferablydetermined by ELISA as described herein. All sera are preferablydepleted of cross-reactive antibodies prior to use in ELISA.

The following description is of a preferred assay method and approach,and is not intended to be limiting to the particular steps (or theirsequence), conditions, reagents and amounts of materials.

Briefly, 200 μl of E. coli lysates (suspended at 500 μg/ml) are coatedonto wells of ELISA plates (Immulon 2, Dynex, Chantilly, Va.) and thewells are blocked with 5% bovine serum albumin (BSA). The serum samples(diluted 1:10 in PBS-Tween-20) are exposed to 8 cycles of absorptionagainst the E. coli lysates. The adsorbed sera are then used in theELISA assays.

Fifty μl of the individual antigens, suspended at 2 μg/ml in coatingbuffer (except for the total culture filtrate proteins which is used at5 μg/ml), are allowed to bind overnight to wells of ELISA plates. After3 washes with PBS (phosphate buffered saline), the wells are blockedwith 7.5% FBS (fetal bovine serum, Hyclone, Logan, Utah) and 2.5% BSA inPBS for 2.5 hr at 37° C. Fifty μl of each serum sample are added perwell at predetermined optimal dilutions (e.g., dilutions of about1:50-1:200). The antigen-antibody binding is allowed to proceed for 90min at 37° C. The plates are washed 6 times with PBS-Tween 20 (0.05%)and 50 μl/well of alkaline phosphatase-conjugated goat anti-human IgG(Zymed, Calif.), diluted 1:2000 in PBS/Tween 20 is added. After 60 minthe plates are washed 6 times with Tris buffered saline (50 mM Tris, 150mM NaCl) and the Gibco BRL Amplification System (Life Technologies,Gaithersburg, Md.) used for development of color. The absorbance is readat 490 nm after stopping the reaction with 50 μl of 0.3M H₂SO₄. Thecutoff in all ELISA assays is determined by using mean absorbance(=Optical Density O.D.) +3 standard deviations (SD) of the negativecontrol group comprising PPD positive and PPD negative healthyindividuals.

The reactivity of the urine samples with the various antigens isdetermined initially with undiluted urine samples as described above.For the urine ELISA, results obtained by the present inventors showedthat the optimal concentration of the culture filtrate proteinpreparation is about 125 μl/well of 4 μg/ml suspension, and for certainproteins, 125 μl/well of about 2 μg/ml. Also, the urine is leftovernight in the antigen coated wells. However, if urine antibody titersof smear-negative and HIV-infected patients are lower than thoseobserved in smear positive patients, it may be necessary to firstconcentrate the urine samples. For concentration, Amicon concentratorswith a molecular weight cut off of 30 kDa is preferred. Concentratedurine samples are evaluated for the presence of antibodies to the abovementioned antigens. Optimal conditions for these assays are determinedreadily. The sensitivity and specificity of antibody detection by use ofone or more of the antigens, with both urine and serum samples is alsoreadily determined.

Vaccines

The present disclosure and Examples prove that human subjects infectedwith Mtb indeed do respond immunologically to early Mtb antigens,including the four surface proteins described more thoroughly herein.Thus the antigens are available to the immune system and areimmunogenic. It is believed that these are stage-specific proteins thatplay some critical role in the microorganisms life cycle at relativelyearly stages of the infectious process. Hence, the vaccine compositionsand methods described herein are designed to augment this immunity, andpreferably, to induce it a stage wherein the bacterial infection can beprevented or curtailed.

The vaccine compositions are particularly useful in preventing Mtbinfection in subjects at high risk for such an infection, as discussedabove. The vaccine compositions and methods are also applicable toveterinary uses for infections with other mycobacterial species such asM. bovis which infects cattle, particularly because these proteins areconserved among mycobacterial species.

Thus, this invention includes a vaccine composition for immunizing asubject against Mtb infection. An Mtb early antigen preferably one ofthe proteins described herein in more detail, is prepared as the activeingredient in a vaccine composition. The vaccine may also comprises oneor more of the proteins described herein, peptides thereof or functionalderivatives as described, or DNA encoding the protein, and apharmaceutically acceptable vehicle or carrier. In one embodiment, thevaccine comprises a fusion protein which includes an Mtb early antigen.The vaccine composition may further comprise an adjuvant or other immunestimulating agent. For use in vaccines, the Mtb early antigen protein orepitope-bearing peptide thereof is preferably produced recombinantly,preferably in prokaryotic cells.

Full length proteins or longer epitope-bearing fragments of the Mtbearly antigen proteins are preferred immunogens, in particular, thosereactive with early antibodies or T cells. If a shorter epitope-bearingfragment, for example containing 20 amino acids or less, is the activeingredient of the vaccine, it is advantageous to couple the peptide toan immunogenic carrier to enhance its immunogenicity. Such couplingtechniques are well known in the art, and include standard chemicalcoupling techniques using linker moieties such as those available fromPierce Chemical Company, Rockford, Ill. Suitable carriers are proteinssuch as keyhole limpet hemocyanin (KLH), E. coli pilin protein k99, BSA,or rotavirus VP6 protein.

Another embodiment is a fusion protein which comprise the Mtb earlyantigen protein or epitope-bearing peptide region fused linearly to anadditional amino acid sequence. Because of the ease with whichrecombinant materials can be manipulated, multiple copies a selectedepitope-bearing region may be included in a single fusion proteinmolecule. Alternatively, several different epitope-bearing regions canbe “mixed and matched” in a single fusion protein.

The active ingredient such, preferably a recombinant product, ispreferably administered as a protein or peptide vaccine. The vaccinecomposition may also comprise a DNA vaccine (e.g., Hoffman, S L et al.,1995, Ann N Y Acad Sci 772:88-94; Donnelly, J J et al., 1997, Annu RevImmunol 15:617-48; Robinson, H L, 1997, Vaccine. 15: 785-787, 1997;Wang, R et al., 1998, Science. 282: 476-480, 1998; Gurunathan, S et al.,2000, Annu Rev Immunol 18:927-74; Restifo, N P et al., 2000, Gene Ther.7: 89-92). The DNA preferably encodes the protein or epitope(s),optionally linked to a protein that promotes expression of the Mtbprotein in the host after immunization. Examples known in the artinclude heat shock protein 70 (HSP70) (Srivastava, P K et al., 1994.Immunogenetics 39:93-8;Suto, R et al., 1995, Science 269:1585-8;Arnold-Schild, D et al., 1999, J Immunol 162:3757-60; Binder, R J etal., 2000, Nature Immunology 2:151-155; Chen, C H et al., 2000, CancerRes 60:1035-42) or translocation proteins such herpesvirus protein VP22(Elliott, G, and O'Hare, P., 1997. Cell 88:223-33; Phelan, A et al.,1998, Nat Biotechnol 16:440-3; Dilber, M S et al., 1999. Gene Ther6:12-21) or domain II of Pseudomonas aeruginosa exotoxin A (ETA) (Jinno,Y et aL, J Biol Chem. 264: 15953-15959, 1989; Siegall, C B et al.,Biochemistry. 30: 7154-7159, 1991; Prior, T I et al., Biochemistry. 31:3555-3559, 1992; Fominaya, J et al., J Biol Chem. 271: 10560-10568,1996; Fominaya, J et al., Gene Ther. 5: 521-530, 1998; Goletz, T J etal., Hum Immunol. 54: 129-136, 1997).

In another embodiment, the vaccine is in the form of a strain ofbacteria (preferably a known “vaccine strain”) which has beengenetically transformed to express the protein or epitope-bearingpeptide. Some known vaccine strains of Salmonella are described below.Salmonella dublin live vaccine strain SL5928 aroA148 fliC(i)::Tn10 andS. typhimurium LB5000 hsdSB121 leu-3121 (Newton S. M. et al., Science1989, 244: 70

A Salmonella strain expressing the Mtb protein or fragment of thisinvention may be constructed using known methods. Thus, a plasmidencoding the protein or peptide. The plasmid may first be selected in anappropriate host, e.g., E. coli strain MC1061. The purified plasmid isthen introduced into S. typhimurium strain LB5000 so that the plasmidDNA is be properly modified for introduction into Salmonella vaccinestrains. Plasmid DNA isolated from LB5000 is introduced into, e.g., S.dublin strain SL5928 by electroporation. Expression of the Mtb proteinor fragment encoded by the plasmid in SL5928 can be verified by Westernblots of bacterial lysates and antibodies specific for the relevantantigen or epitope.

The active ingredient, or mixture of active ingredients, in protein orpeptide vaccine composition is formulated conventionally using methodswell-known for formulation of such vaccines. The active ingredient isgenerally dissolved or suspended in an acceptable carrier such asphosphate buffered saline. Vaccine compositions may include animmunostimulant or adjuvant such as complete or incomplete Freund'sadjuvant, aluminum hydroxide, liposomes, beads such as latex or goldbeads, ISCOMs, and the like. For example, 0.5 ml of Freund's completeadjuvant or a synthetic adjuvant with less undesirable side effects isused for intramuscular or subcutaneous injections, preferably for allinitial immunizations; this can be followed with Freund's incompleteadjuvant for booster injections. General methods to prepare vaccines aredescribed in Remington's Pharmaceutical Science; Mack Publishing CompanyEaston, Pa. (latest edition).

Liposomes are pharmaceutical compositions in which the active protein iscontained either dispersed or variously present in corpuscles consistingof aqueous concentric layers adherent to lipidic layers. The activeprotein is preferably present in the aqueous layer and in the lipidiclayer, inside or outside, or, in any event, in the non-homogeneoussystem generally known as a liposomic suspension. The hydrophobic layer,or lipidic layer, generally, but not exclusively, comprisesphospholipids such as lecithin and sphingomyelin, steroids such ascholesterol, more or less ionic surface active substances such asdicetylphosphate, stearylamine or phosphatidic acid, and/or othermaterials of a hydrophobic nature. Adjuvants, including liposomes, arediscussed in the following references, incorporated herein by reference:Gregoriades, G. et al., Immunological Adjuvants and Vaccines, PlenumPress, New York, 1989 Michalek, S. M. et al., “Liposomes as OralAdjuvants,” Curr. Top. Microbiol. Immunol. 146:51-58 (1989).

The vaccine compositions preferably contain (1) an effective amount ofthe active ingredient, that is, the protein or peptide together with (2)a suitable amount of a carrier molecule or, optionally a carriervehicle, and, if desired, (3) preservatives, buffers, and the like.Descriptions of vaccine formulations are found in Voller, A. et al., NewTrends and Developments in Vaccines, University Park Press, Baltimore,Md. (1978).

As with all immunogenic compositions for eliciting antibodies orcell-mediated immunity, the immunogenically effective amounts of theproteins or peptides or other vaccine compositions of the invention mustbe determined empirically. Factors to be considered include theimmunogenicity of the native peptide, whether or not the peptide will becomplexed with or covalently attached to an adjuvant or carrier proteinor other carrier and the route of administration for the composition,i.e., intravenous, intramuscular, subcutaneous, etc., and the number ofimmunizing doses to be administered. Such factors are known in thevaccine art, and it is well within the skill of the immunologists tomake such determinations without undue experimentation.

The vaccines are administered as is generally understood in the art.Ordinarily, systemic administration is by injection; however, othereffective means of administration are known. With suitable formulation,peptide vaccines may be administered across the mucus membrane usingpenetrants such as bile salts or fusidic acids in combination, usually,with a surfactant. Transcutaneous administration of peptides is alsoknown. Oral formulations can also be used. Dosage levels depend on themode of administration, the nature of the subject, and the nature ofcarrier/adjuvant formulation. Preferably, an effective amount of theprotein or peptide is between about 0.01 μg/kg-1 mg/kg body weight.Subjects may be immunized systemically by injection or orally byfeeding, e.g., in the case of vaccine strains of bacteria, 10⁸-10¹⁰bacteria on one or multiple occasions. In general, multipleadministrations of the vaccine in a standard immunization protocol areused, as is standard in the art. For example, the vaccines can beadministered at approximately two to six week intervals, preferablymonthly, for a period of from one to four inoculations in order toprovide protection.

Vaccination with the vaccine composition will result in an immuneresponse, either or both of an antibody response and a cell-mediatedresponse, , which will block one or more steps in the Mtb bacterium'sinfective cycle, preferably the steps of binding to and entry into hostcells in which it grows.

T Cell Responses

Human (or animal model) peripheral blood lymphocytes (PBL) orlymphocytes from another source (e.g., lymph node) are incubated incomplete culture medium (as are well known in the art) at appropriatecell concentrations. A preferred medium is RPMI 1640, supplemented with10% (vol/vol) fetal bovine serum, 50 units/ml penicillin/ streptomycin,2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM nonessential amino acids)

Cells are stimulated for an appropriate period, e.g., 2-4 days with anMtb protein or peptide fragment thereof antigen at concentrationsreadily ascertainable by those of skill in the art. Interleukin 2 (IL-2)can be added to promote expansion of antigen-specific cells if it isdesired to generate antigen-specific lines or clones.

T cells from a PPD+ normal individual or from a patient being tested arecultured with varying concentrations of an Mtb protein or peptide beingevaluated for its T cell stimulatory capacity. T cell reactivity ismeasured in any of a number of conventional assays, for example T cellproliferation which can be measured by radiolabeled thymidine oriododeoxyuridine, or by colorimetric assay of cell number.Alternatively, stimulation of T cell activity can be measured bysecretion of cytokines or by ELISPOT assays that enumerate cytokinesecreting cells.

The enzyme-linked immunospot (ELISPOT) assay described (e.g. Miyahira, Yet al., J Immunol Methods. 181: 45-54, 1995) utilizes 96-well filtrationplates (Millipore, Bedford, Mass.) coated with about 10 μg/ml of anantibody (commercially available) specific for a cytokine being assayedin 50 μl PBS. After overnight incubation at 4° C., the wells are washedand blocked with culture medium containing 10% fetal bovine serum.Different concentrations of fresh isolated lymphocytes being assayedstarting from 1×10⁶/well, are added to the well along with 15international units/ml interleukin-2 (IL-2). Cells are incubated at 37°C. for 24 hours either with or without a stimulatory amount of the Mtbprotein or peptide thereof. After culture, the plate is washed and thenfollowed by incubation with 5 μg/ml biotinylated antibody specific forthe cytokine being assayed (e.g., IFN-γ) in 50 μl in PBS at 4° C.overnight. After washing six times, 1.25 μg/ml avidin-alkalinephosphatase (Sigma, St. Louis, Mo.) in 50 μl PBS are added and incubatedfor 2 hours at room temperature. After washing, spots are developed byadding 50 μl BCIP/NBT solution (Boehringer Mannheim, Indianapolis, Ind.)and incubated at room temperature for 1 hr. The spots are counted usinga dissecting microscope.

Intracytoplasmic Cytokine Staining and Flow Cytometry Analysis

Lymphocytes are incubated either with the Mtb protein or peptide at anappropriate concentration for about 20 hours. Golgistop (Pharmingen, SanDiego, Calif.) is added 6 hours before harvesting the cells from theculture. Cells are then washed once in an appropriate buffer for flowcytometry and stained with appropriately labeled (e.g.,phycoerythrin-conjugated) anti CD8 or anti-CD4 antibody. Cells aresubjected to intracellular cytokine staining using the Cytofix/Cytopermkit according to the manufacturer's instructions (e.g., fromPharmingen). FITC-conjugated anti-cytokine antibodies and theimmunoglobulin isotype control antibody are used. Analysis was done on aflow cytometer

ELISA for Cytokines

Lymphocytes (e.g., 4×10⁶) are obtained from subjects or from culture andare incubated in culture medium with Mtb protein or peptide in a totalvolume of 2 ml of medium in a 24-well tissue culture plate for 72 hours.The supernatants are harvested and assayed for the presence of cytokine,e.g., IFN-γ or IL12 or IL18 using commercial ELISA kits according tomanufacturer's protocol.

Antibody Responses to Mt

The humoral responses to Mtb in TB patients have been the subject ofinvestigation for several decades, primarily for the purpose of devisingserodiagnosis for TB (reviewed in Grange, J M, 1984, Adv Tuberc Res.21:1-78). The earlier studies of humoral responses in TB patients weremostly based on use of crude mixtures of antigens like PPD, bacterialsonicates, Ag A60 etc. These antigen preparations providedunsatisfactory results, because although a majority of TB patients wereantibody positive, often-healthy individuals also had antibodies thatshowed cross-reactivity with these preparations. A variety ofapproaches, both biochemical and recombinant, were then used bydifferent labs to obtain individual, purified antigens of Mtb (Young, DB et al., Mol. Microbiol. 6:133-145). Studies of purified antigensshowed that many of the Mtb antigens are conserved, prokaryotic proteinswhich have significant homology with analogous proteins in othermycobacterial and non-mycobacterial organisms (the 65 kDa GroEL, 70 kDaDNA K, 47 kDa elongation factor Tu, 44 kDa Pst A homolog, 40 kDaL-alanine dehydrogenase, 23 kDa superoxide dismutase, 23 kDa outermembrane protein, 14 kDa GroES, enzymes of metabolic pathways etc (Younget al., supra). Studies also showed that healthy individuals often haveantibodies to epitopes on conserved regions of such ubiquitousprokaryotic proteins, resulting in the observed cross-reactivity of thehealthy sera with mycobacterial antigens. Some of the purifiedmycobacterial antigens were evaluated for their use in serodiagnosis ofTB, and one of them, a 38-kDa protein provided promising results. Thisantigen provided very high specificity (>98%). However, extensivestudies with the 38 kDa protein in different populations showed thatanti-38 kDa antibodies are present only in individuals with chronic,recurrent, cavitary TB, limiting its utility in diagnosis of TB(Bothamley, G H et al., 1992, Thorax. 47:270-275; Daniels, T M, 1996, p.223-231. In W. R. Rom and S. Garay (ed.), In: Tuberculosis. Little,Brown and Company, Inc, Boston, Mass.).

Most of the purified antigens that were evaluated for their utility forserodiagnosis were either proteins that were immunodominant in mice thatwere immunized with killed preparations/sonicates of Mtb or BCG for thepurpose of producing monoclonal antibodies (Engers, H D et al., 1986,Infect. Immun. 51:718-720), or were antigens that were relatively easyto purify by biochemical procedures (Sada, E et al., 1990, J. ClinMicrobiol. 28:2587-2590; Sada, E et al., 1990, J. Infec. Dis.162:928-931). Based on the rationale that there may be differences inantigens expressed by in vivo replicating bacteria, and inactivatedantigen preparations, our approach was to perform a direct analysis ofantibody responses in patients with active TB.

We developed a unique approach to address the issue of cross-reactivitydescribed above, and have provided evidence that adsorption of sera withlysates of E. coli, which contain many of the ubiquitous prokaryoticproteins, results in significant depletion of the cross-reactiveantibodies. Using cross-reactive antibody depleted sera, we havesystematically dissected the antibody responses of both HIV-infected andnon-HIV TB patients at different stages of disease progression. Ourstudies show that the culture filtrate antigens are targets of humoralresponses during active infection in humans, and that antibodies to theculture filtrate proteins are present in individuals with active TB, andnot in PPD positive healthy individuals. We have defined the repertoireof antigens in culture filtrates of Mtb that elicit antibodies in TBpatients by using 2-D fractionated proteins and immunoblotting. Ourstudies show that of the >100 different proteins released byextracellularly growing Mtb, antibodies to only a small number ofproteins (18 antigens) are present in non-HIV TB patients withnon-cavitary disease. HIV-infected TB patients, a majority of whom alsohas non-cavitary disease, have antibodies to the same small subset ofculture filtrate antigens. In contrast, a majority of the advancedcavitary TB patients have antibodies to the above 18 antigens, andseveral additional antigens. These studies make several importantpoints.

First, the reactivity of TB sera with the culture filtrate proteins, andthe lack of reactivity of sera from PPD positive healthy individualswith the same antigens suggest that antibodies to these antigens areassociated with active TB infection.

Second, the sera from TB patients with only a minority of the culturefiltrate proteins of Mtb suggests that many of the culture filtrateproteins may not be expressed in significant amounts by the in vivoreplicating bacteria.

Third, the differences in the antigen profiles recognized by thenon-cavitary and cavitary TB patients suggests that the local milieu(intracellular vs. extracellular, extent of liquification, cavitation,etc.) in which the in vivo bacteria exist affects the antigen profilesexpressed. We also showed that 3 of these 26 culture filtrate proteins,identified on the basis of their reactivity with TB sera, are useful forserodiagnosis for pulmonary TB.

Although the culture filtrates have yielded important molecules fordiagnosis of TB, they would only contain antigens that are expressed byMtb replicating in vitro in bacteriological media. Other antigens thatare expressed by the bacteria during in vivo growth may be poorlyexpressed or even absent in these preparations. Recently at least 3antigens of Mtb that are expressed/upregulated in intracellularconditions, or in vivo, or in granulomas have been reported. In fact,the ability of bacteria to respond to environmental changes is a keyfeature in their ability to survive, and differential expression ofproteins in vivo and in vitro, and of different proteins duringdifferent stages of disease progression has been reported for severalpathogens. The importance of the effect of the immune system componentson bacterial survival and growth is also emphasized by the opportunisticpathogens that cause disease only in individuals with compromised immunesystems. A multitude of factors—cytokine levels, iron availability, pH,osmolarity etc can affect the gene expression, and therefore the geneproducts, expressed by the bacteria in vivo. The search for moleculesthat may be useful for early diagnosis of TB should ideally be focusedon antigens expressed by the in vivo bacteria during the earliest stagesof infection. Yet, most current studies have focused either on antigensexpressed by the bacteria growing in vitro in bacteriological media oron antigens recognized by sera of patients with clinical disease.

Recent experiments with increasing doses of BCG as a vaccine in miceshowed that regardless of the route of immunization, high doses of BCGactivated Th2 responses (Power, C A et al.., 1998, Infect. Immun.66:5743-5750). Other studies in which humoral, cellular and protectiveimmune responses were monitored in individual animals immunized with DNAvaccines encoding several different Mtb antigens have shown thatantibody concentrations reflected the levels of antigenic expression(Li, Z et al., 1999, Infect. Immun. 67:4780-4786). These studiessuggested that the presence of antibodies to any protein can serve as anexcellent marker of expression of high levels of that protein in vivo.Since the exact in vivo environment is impossible to replicate in vitroor in culture, the studies described herein are based on usingantibodies as tools to identify antigens that are expressed by Mtb invivo during the early stages of disease progression. Other investigators(Amara, R R et al., 1996, Infect. Immun. 64:3765-3771) used antibodiesfrom TB patients to identify antigens of Mtb expressed in vivo and haveidentified several novel antigens. However, these antigens are thosethat were recognized by sera from patients with chronic,culture-positive TB, and represent antigens expressed in an environmentwhere there is marked caseous necrosis, liquification of caseousmaterial and cavity formation—an environment that allows extensiveextracellular replication of the in vivo bacteria. In contrast, prior tothe development of extensive pulmonary lesions, the bacteria arebelieved to be primarily intracellular, an environment that is differentfrom the cavity environment.

Whether the same antigens are expressed by the in vivo Mtb during theearly and the late stages of active disease is not known. Our ownstudies with non-cavitary and cavitary TB patients have shown thatcavitary patients have antibodies to several antigens that are notrecognized in non-cavitary patients, suggesting that the antigen profileexpressed in vivo is altered with disease progression as the environmentin which the bacteria survive changes. Thus, sera from advanced TBpatients are likely to be enriched for antibodies to antigens expressedby the bacteria replicating extracellularly in cavitary lesions. Thepresent invention focuses on identifying and obtaining antigens of Mtbthat are expressed in vivo, and elicit immune responses during theearly, pre-clinical stages of an active infection with Mtb. Sera frompatients with active, early TB cannot be obtained from humans becausethe lifetime risk of a latently infected individual (PPD positive)developing active clinical TB is so small that the size of the cohort ofPPD positive individuals that would have to be studied for theirlifetimes, to identify some individuals who may develop disease is notpossible at the practical level. Yet, the antigens expressed during theearly stages of disease may play an important role in determining theoutcome of infection and may prove useful in serodiagnostic assays fordiagnosis of early, active infection with Mtb.

To identify the antigens expressed by in vivo replicating Mtb during theearly stages of disease progression, studies were done with antibodiesfrom guinea pigs infected by virulent airborne organisms. The guinea pigmodel is considered especially relevant to humans, clinically,immunologically and pathologically (Smith, D W et al., 1989. Reviews ofInfect. Dis. 2:s385-s393). In contrast to the mouse and rat, but likethe humans, guinea pigs are susceptible to low doses of airborne Mtb,have a strong cutaneous DTH to tuberculin, and display langans giantcells and caseation in pulmonary lesions. Earlier studies of the courseof infection in guinea pigs following low dose, pulmonary infection withMtb have revealed that the mycobacteria replicate exponentially in thelungs during the first 3-21 days post-infection in the lungs (Smith, D Wet al., 1970, Am Rev. Respir Dis 102:937-949). Dissemination via thelymphatics to the lymph nodes draining the lung fields occurs at about8-10 days post-infection, with organisms reaching the spleen via thebloodstream between 14 and 20 days. Within 4 weeks following initiationof the pulmonary infection, there is seeding of mycobacteria intoso-called secondary foci throughout the lungs via hematogenousdissemination. Clinical signs of TB in these guinea pigs, such as weightloss and respiratory distress, usually occur at 8-10 weekspost-infection, with mortality observed at 14-18 weeks (Wiegeshaus, E Het al., 1970, Am. Rev. Respir. Dis. 102:422-429). For studies ofantigens that are expressed during the early stages of bacterialreplication and dissemination by in vivo growing bacteria, serum sampleswere obtained from guinea pigs infected with airborne virulent Mtb.These sera, obtained at 1, 3, 4, 5 and 6 weeks post infection wereprovided by Dr. David McMurray, Texas A & M University. One serumsample, obtained from a guinea pig 8 weeks post infection was obtainedfrom Dr. John Belisle, Colorado State University. Thus, the period oftime during which the sera used in this study were collected reflectsthe early post-infection period during which rapid bacillarymultiplication and dissemination is known to occur in the lung andelsewhere. These sera would contain antibodies directed against antigensexpressed by the bacteria replicating and disseminating in vivo and weretherefore used to screen a λgt11 expression library of Mtb to obtain theclones expressing these antigens (Young, R A et al., 1985, Proc. Natl.Acad. Sci. USA. 82:2583-2587). These are the proteins expressed duringthe early stages of disease progression by in vivo growing bacteria. Ourinitial studies showed that these antigens were also recognized duringinfection with Mtb in humans. These antigens, therefore, are useful asdiagnostic reagents

The following examples are directed to the discovery of four novelrepetitive proteins that are immunodominant as Mtb antigens during earlytuberculosis

To identify antigens of Mtb expressed during early TB, rabbits wereinfected by aerosols of Mtb H37Rv or a clinical isolate CDC1551, andbled 5 weeks post-infection. These sera were used to immunoscreen aλgt11 genomic DNA expression library of Mtb. Seven positive clones wereobtained, five of which were sequenced. Clones AD1 and AD2 expressoverlapping portions of C-terminal of the protein PirG (Rv 3810). Theproduct of the PirG has previously been shown to be a cell surfaceexposed protein associated with virulence of Mtb (Berthet, F.-X. et al.,1998, Science. 282:759-762). Clones AD9, AD10 and AD16 express theC-terminal portion of a PE_PGRS (Rv3367) glycine rich protein; a prolineand threonine rich protein PTRP (Rv 0538) and the protein MtrA (Rv3246c) respectively. Three of the proteins, PirG, PE_PGRS and PTRP arerepetitive proteins, and have multiple tandem repeats of unique aminoacid motifs while the fourth protein, MtrA is a response regulator of aputative two component signal transduction system mtrA-mtrB of Mtb,which has been shown to be upregulated on intracellular entry andresidence of Mtb in macrophages (44). All four antigens were recognizedby pooled sera from cavitary TB patients confirming their in vivoexpression in human TB. Three of the antigens, (PE_PGRS, PTRP and MtrA)were also reactive with sera from non-cavitary TB and HIV pre-TBindividuals suggesting that these proteins are expressed in vivo earlyduring an active infection.

Studies performed by the present inventors' laboratory identifying theantigens in culture filtrates of Mtb recognized by antibodies fromnon-cavitary and/or cavitary TB patients are published (describedsupra). Studies with sera from the aerosol-infected guinea pigs arepresented below in Examples I-V. A list of references following ExamplesI-V contains the references cited by parenthetical number in theseExamples.

Subsequent Examples VI-XIII are directed to the discovery of four novelrepetitive proteins that are immunodominant as Mtb antigens during earlytuberculosis To identify antigens of Mtb expressed during early TB,rabbits were infected by aerosols of Mtb H37Rv or a clinical isolateCDC1551, and bled 5 weeks post-infection. These sera were used toimmunoscreen a λgt11 genomic DNA expression library of Mtb. Sevenpositive clones were obtained, five of which were sequenced. Clones AD1and AD2 express overlapping portions of C-terminal of the protein PirG(Rv 3810). The product of the PirG has previously been shown to be acell surface exposed protein associated with virulence of Mtb (Berthet,F.-X. et al., 1998, Science. 282:759-762). Clones AD9, AD10 and AD16express the C-terminal portion of a PE_PGRS (Rv3367) glycine richprotein; a proline and threonine rich protein PTRP (Rv 0538) and theprotein MtrA (Rv 3246c) respectively. Three of the proteins, PirG,PE_PGRS and PTRP are repetitive proteins, and have multiple tandemrepeats of unique amino acid motifs while the fourth protein, MtrA is aresponse regulator of a putative two component signal transductionsystem mtrA-mtrB of Mtb, which has been shown to be upregulated onintracellular entry and residence of Mtb in macrophages (Via, L et al.,1996, J. Bacteriology. 178:3314-21). All four antigens were recognizedby pooled sera from cavitary TB patients confirming their in vivoexpression in human TB. Three of the antigens, (PE_PGRS, PTRP and MtrA)were also reactive with sera from non-cavitary TB and HIV pre-TBindividuals suggesting that these proteins are expressed in vivo earlyduring an active infection.

EXAMPLE I Examination of Sera of Infected Guinea Pigs

Serum samples: Sera obtained from 2 uninfected guinea pigs and 20 guineapigs infected with 4-10 cfu, airborne, virulent Mtb H37Rv, and bled at1,3,4,5, and 6 weeks post-infection, were provided by Dr. DavidMcMurray. Serum from one guinea pig infected for 8 weeks was obtainedfrom Dr. John Belisle. A serum pool containing one serum each fromguinea pigs bled 1, 3-6 weeks post-infection and 8 weeks post infectionsample was absorbed against an E. coli lysate and used at a dilution of1:100 for probing the western blots (described below) and forimmunoscreening the expression library.

Reactivity of guinea-pig serum pool with antigens of Mtb: The reactivityof the above serum pool was assessed with the following antigenpreparations of Mtb (provided by Dr. John Belisle, Colorado StateUniversity) by western blot analyses:

-   a) Bacterial cell sonicate (CS): the cell pellet of organisms    harvested by centrifugation, sonicated extensively, and subjected to    high speed centrifugation to get rid of the cell-wall fragments.    This preparation contains primarily cytoplasmic proteins of Mtb.-   b) SDS-soluble cell-wall proteins (SDS-CW): the proteins associated    with the bacterial cell wall, extracted as described in (Laal, S et    al., 1997, J. Infect. Dis. 176:133-143).-   c) Lipoarabinomannan-free culture filtrate proteins (LAM-free CFP)    from log phase Mtb: This preparation contains the proteins secreted    by bacteria replicating in vitro (in bacteriological media)    (Sonnenberg, M G et al., 1997, Infect. Immun. 65:4515-4524).

The preparation of these antigens has been described before (Laal etal., supra). Western blots prepared after SDS-PA gel fractionation ofthese antigens were probed with the guinea-pig serum pool at a dilutionof 1:100. The filters were washed, exposed to 1:1000 dilution ofalkaline phosphatase conjugated anti-guinea-pig IgG, washed anddeveloped with BCIP-NBT substrate.

Only the serum pool from Mtb infected guinea pigs reacted strongly witha 40 kDa protein present in the total CS of Mtb (FIG. 1, lane 3). Weakerreactivity was also seen with a doublet at 50-52 kDa (lane 3). Severalother proteins reacted strongly with the infected guinea pig serum pool(lane 3) and weakly with the uninfected guinea pig serum pool (lane 2).A 40 kDa protein was also identified only by the serum pool frominfected animals in the SDS-CW preparation, as were several weaklyreactive bands ranging from 48-103 kDa (lane 5). None of the antigens inthe LAM-free CFP preparation showed any specific reactivity with theserum pool from the infected animals.

Screening of the Mtb λgt11 expression library with guinea pig sera: Toobtain the antigens recognized by the sera from the aerosol infectedguinea-pigs, the above serum pool was used to screen a λgt11 expressionlibrary of Mtb DNA (World Health Organization (Young, R A et al., 1985,Proc. Natl. Acad. Sci. USA. 82:2583-2587). The details of the libraryand the methods for screening are described in the Experimental Designsection. Several recombinant phages, 10 of which could be cloned byseveral rounds of screening, were obtained. These are referred to as gsrI-3, and I-6, which were obtained during preliminary screening, and gsrII-1, II-2, II-3, II-4, II-6, II-14, II-15 and II-20 which were obtainedduring the second round of screening.

Characterization of the recombinant proteins: Lysogens of the gsr-cloneswere established in E. coli Y1089. Single colonies from lysogens wereused to obtain the recombinant proteins (35). The E. coli lysatescontaining the recombinant proteins were fractionated on 10% SDS-PA gelsand electroblotted onto nitrocellulose membranes. Lysates from severalindividual colonies from each of the lysogens were tested. The blotswere probed with the guinea-pig serum pool and separate blots were alsoprobed with a commercially available murine mAb against β-galactosidase.Lysates from E. coli Y1089 alone were used as controls. FIGS. 2 a-d and3 a show the results of these experiments. β-gal-fusion proteins werepresent in the lysates of lysogens from all the 10 recombinant phagesobtained from the library.

Further studies were performed to confirm that the reactivity of thesera from the infected animals with the fusion proteins was with themycobacterial fragment, and not the β-galactosidase portion of thefusion protein (FIG. 3 b). E coli Y 1089 was lysogenized with the emptyλgt11 vector, induced to express the β-galactosidase, and the blotsprobed with the anti-β-gal antibody, or the guinea-pig serum pool. Onlythe former antibody showed significant reactivity with theβ-galactosidase band in the induced lysate (FIG. 3 b).

Cross hybridization between the 10 clones: DNA from {fraction (9/10)}gsr clones was isolated by the commercial Wizard L Preps DNAPurification system (Promega), and digested with EcoR1 to determine theinsert size. To determine if some of the gsr clones were related to theothers, the insert DNA from {fraction (9/10)} clones was isolated, andlabeled with ³²P by a random priming DNA labeling. The DNA from all thegsr clones (except gsr II-14 and II-20) was digested with EcoR1,transferred from the agarose gels to a Nytran Plus filters (Schleicher &Schuell, Keene, N.H.) and the filters subjected to Southern blotanalysis using the labeled insert DNA from the gsr clones. Thehybridization pattern revealed that insert DNA from clones gsr I-3, II-3and II-6 cross hybridized, while inserts of gsr I-6, II-1, II-2, II-4and II-15 hybridized only with the parent clone. The status of clonesgsr II-14 and gsr II-20 remains to be determined. Thus, at least six ofthe eight clones are independent clones. Clones gsr I-6, gsr II-1 andgsr II-2 were randomly selected for initial studies.

DNA Sequence Analyses: λDNA from clones gsr I-6, II-1 and II-2 wasdigested with EcoR1 and the insert subcloned into vector pGEMEX-1(Promega) whose reading frame at the EcoR1 cloning site is identical toλgt11. Competent E. coli JM 109 cells were transformed with therecombinant plasmid (pGEMEX plus insert from gsr clones). Plasmid DNAwas isolated using Wizard Plus Minipreps (Promega), and used forautomated sequencing with SP6 and T3 promoter specific primers flankingthe multiple cloning site in the pGEMEX-1 followed by primer walking.The sequencing was performed by the NYU Medical Center core sequencingfacility. The nucleotide sequences obtained were used in homologysearches using the NCBI BLAST search (1). Nucleotide sequences for all 3clones shared homology to known Mtb sequences.

DNA sequence analyses of clones gsr I-6 (0.7 kb) and gsr II-2 (2.1 kb)showed 98% and 99% homology respectively to different regions of thesame gene (Mtb cosmid MTV004.03c: nu 4314-13787, FIG. 4 a). Clone gsrI-6 (nu 1-720) and gsr II-2 (nu 1-1903) showed homology to nu 5870-6590and nu 2573-4476 of MTV004 respectively. A DNA fragment (152 bp) at the3′ end of gsr II-2 showed only 60% homology to MTV004 (and several otherregions on the mycobacterial genome) indicating that scrambling mighthave occurred during construction of the library. The gene product ofMTV004.03 is a 3175 aa (309 kDa) member of the recently described PPEfamily of Gly-, Ala-, Asn-rich proteins (FIG. 4 b). The peptidesexpressed in gsr I-6 and gsr II-2 represent aa 2400-2639 and 3104-3157respectively in the C-terminal half of the MTV004.03 gene product (FIG.4 b).

The PPE family of acidic proteins was described recently when the genomesequence of Mtb was analyzed (5). This family of proteins has 68members, all of who possess a conserved N-terminal domain of 180 aa, anda ProProGlu (PPE) motif at positions 7,8 and 9. Based on thecharacteristics of the C-terminal portion, the PPE proteins fall into 3groups, one of which (MPTR family) is characterized by the presence ofmultiple copies of the AsnXGlyXGlyXAsnXGly motif (5). Analyses of theprotein encoded by MTV004.03 by FINDPATTERN revealed the presence of 65tandem copies of this motif spanning the entire length of the protein in5 clusters. The motif is repeated 6 times in the region encoded by clonegsr I-6. The sequence of gsr II-2 did not contain this motif (FIG. 4 b).

Clone gsr II-1 (2.14 kb) nucleotide sequence showed homology to Mtbcosmid Y336 (A# Z95586) region 22232-24371. Nucleotides 1-819 (273 aa)of gsr II-1 showed 96% homology to nu 22232-23050 (ORF MTCY336.28) ofMTCY336 (FIG. 5 a). This clone also showed homology to the RD3 region ofM. bovis (24) which is represented by sequences with accession numbersU35017 (MBDR3S1) and U35018 (MBDR3S2). Nucleotides 1-819 of gsr II-1showed 99% homology to nu 8370-9189 (ORF 3H) of MBDR3S1. The orientationof the sequence was established by restriction analysis. The geneproduct of MTCY336.28 is a 50 kDa protein with unknown function (FIG. 5b). The RD3 region has been described to be present in Mtb Erdman and M.bovis, but absent from BCG and BCG substrains Connaught, Pasteur andBrazil.

Reactivity of the fusion proteins with sera from guinea-pigs with earlyTB: To determine the earliest time point post-infection at which theseantigens are recognized by the infected animals, reactivity of therecombinant proteins of clones gsr I-6, II-1 and II-2 with individualguinea pig sera were tested. Western blots prepared from lysate fromclone gsr I-6 were probed with the individual sera that were included inthe pool used for immunoscreening of the library. The fusion proteinband was strongly reactive with the sera obtained 1,3 and 4 weekspost-infection, and weakly reactive with the sera obtained 5 and 6 weekspost-infection. A pool of these sera failed to identify a correspondingsized protein in the control E. coli lysate. (FIG. 6 a).

The reactivity of lysate from clone gsr II-1 was evaluated with serumsamples from 4 animals each, obtained at 1 and 3 weeks post-infection.(FIG. 6 b). Three of the 4 sera at 1 week post-infection and 3 of the 4sera at 4 weeks post-infection (total 6 out of 8 animals) showedreactivity with the fusion protein in the gsr II-1 lysate. The same serawere also tested for reactivity with the lysate of clone gsr II-2 (FIG.6 c). Three of the 4 sera obtained 1 week post-infection and all 4samples at weeks 3 post-infection (total 7 out of 8 animals) showedreactivity with the fusion protein. Sera from 2 uninfected guinea pigsshowed no corresponding reactivity with either of the lysates. Thus, theantigens encoded by all 3 clones tested showed reactivity with serumsamples from animals bled during the early stages of disease progression(1-3 weeks post-infection).

The same 8 sera from weeks 1 and 3 post infection were evaluated forreactivity with the 3 antigen preparations: (CS, SDS-CW and LAM-freeCFP). In contrast to the results obtained with the serum pool comprisingof sera from animals bled 1-8 weeks post-infection (used for libraryscreening, FIG. 1), all 8 sera from animals bled 1 and 3 weekspost-infection failed to show reactivity with any antigen in the abovepreparations. However, some of the sera obtained 4-6 weekspost-infection, and the serum obtained 8 weeks post-infection showedreactivity with the three antigen preparations (data not shown).

Validation of use the of guinea pig as model system for human TB: Thereare no markers to identify humans who have an active but subclinicalinfection with Mtb, which may be considered equivalent to the first fewweeks post-infection stage of aerosol infected guinea pigs. Therefore,in order to validate the use of these proteins in studies of humanimmune responses, the following studies have been done:

a. Comparison of antibody reactivity of tuberculous guinea pigs andpulmonary TB patients: Sera from 2 guinea pigs who were infected withaerosolized, virulent Mtb, and bled at 15 weeks post-infection, whenthey have advanced TB, were obtained from Dr. McMurray. The reactivityof these sera with the culture filtrate and cell-wall associatedproteins of Mtb was assessed, and compared to reactivity of sera from 4patients with confirmed pulmonary TB. Culture filtrate proteins andcell-wall associated proteins of Mtb were fractionated on SDS-PA gels,and the western blots probed with the human TB and guinea pig TB sera ata dilution of 1:100. As seen in FIG. 7, the protein bands in the twoantigen preparations recognized by the human and animal TB sera wereremarkably similar. Thus, at the advanced stage of active infection,tuberculous humans and guinea pigs have antibodies to the same antigens.While sera from only four TB patients have been studied to date, thefinding that 4/4 patients showed similar reactivity to the reactivityobserved with sera from tuberculous guinea pigs suggests that ourhypothesis that the guinea pig is adequate as a model system relevant tohuman TB is tenable and worthy of further studies.

b. Reactivity of human TB sera with gsr recombinant proteins: Thereactivity of the fusion proteins from seven gsr clones with a pool ofsera from 6 PPD positive, healthy individuals, and a pool from 6 TBpatients was evaluated. (FIG. 8). Fusion proteins expressed in 5/7clones were reactive with the pooled TB sera, but not the PPD pool.These results suggest that human TB patients have antibodies to thefusion proteins recognized by the guinea-pig sera. Individual sera fromvarious cohorts have not been evaluated for reactivity with the fusionproteins because each of these β-gal-fusion proteins contains only asmall fragment of the original mycobacterial protein, and these smallfragments may not account for the immune response to this protein inevery diseased individual. For example, the mycobacterial DNA fragmentin gsr I-6 expresses only 240 aa of the total 3147 aa long PPE protein,and gsr II-2 expressed only 74 aa of the same protein. Thus, the twoclones express ˜7.5% and 1.7% respectively, of the parent molecule.Positive reactivity of any fusion protein with human TB sera wouldindicate that the antigen is relevant to human TB. However, the smallfragment of the mycobacterial antigen in the fusion protein lacks mostof the regions/epitopes/conformations expressed by the parent molecules,and to which the patients are exposed. The absence of a significantportion of the original protein could provide false negative resultswhen studying individual sera. Moreover, serum samples, especially thepre-clinical TB sera, are available in small amounts (100-200 μl). Thus,it would be unwise and premature to use these valuable sera to testreactivity with the fusion proteins. These sera are being saved fortesting with the complete recombinant proteins once they are produced(EXAMPLE 3). This is the reason that more preliminary data with theserum panels available has not been generated. Nevertheless, thereactivity of the fusion protein in gsr1-6 was tested with individualsera from 3 PPD positive individuals, and 4 TB patients. Although thenumber of individuals tested is small, the reactivity of sera fromseveral TB patients confirms that this protein is recognized during TBin humans (FIG. 9).

In order to determine if these fusion proteins are also recognizedduring the early stages of disease progression, the reactivity of thefusion proteins from seven gsr clones with a pool of sera from 6 PPDpositive, healthy individuals, and a pool from 6 HIV-pre TB sera wasalso evaluated. These pre-TB sera are retrospective, stored serumsamples that were obtained from HIV-infected individuals prior to theirdeveloping clinical TB (cohort described in EXAMPLE 4) and represent theearliest stage of TB that can be diagnosed in humans. Fusion proteinsfrom 2 clones (PPE protein encoded by gsr II-2 and the fusion proteinencoded by clone gsrII-4) showed reactivity with the pool of the pre-TBsera, and not the pool of PPD control sera Whether the protein expressedby the remaining clones are genuinely not recognized by the pre-TB seraremains to be confirmed since this experiment was done at one serumdilution (1:200) (HIV-infected patients may have lower titers ofantibodies) and will be repeated with more concenrated sera, and withcomplete proteins to confirm the negative results. The volumes of pre-TBsera available are very small (100-200 μl), and studies with individualpre-TB sera are performed only after the full-length proteins areexpressed and purified. Such well defined sera are difficult to obtain,and we know of no other cohort that exists.

Ongoing studies with the PPE protein: In order to determine thedistribution of the PPE protein encoded by gsr I-6, genomic DNA from MtbH37Rv, Mtb H37Ra, Mtb Erdman, clinical isolates CSU 11, CSU 17, CSU 19,CSU 22, CSU 25, CSU 26 and CSU 27, M. bovis, M. bovis BCG, M. africanum,M. microti, M. smegmatis, M. vaccae, M. phlei, M. chelonae, and M.xenopii was digested with Eco R1 and southern blofted. A PCR productcorresponding to a 481 bp sequence of gene MTV004.03c was used to probethe southern blot. The gene for this PPE protein is present in allmembers of the TB complex, and all the clinical isolates tested but notin the non-TB mycobacterial species tested (FIG. 10). Currently moreclinical isolates and non-TB mycobacterial species are being evaluatedto confirm the specificity of this gene. This is continued and expandedas part of EXAMPLE 2.

Several unsuccessful efforts have been made to detect the 309 kDaprotein in the culture filtrates or cell wall preparations or sonicatesof Mtb. Either this protein is not expressed/very poorly expressedduring in vitro growth of Mtb, or is expressed by the bacteria butdestroyed by the purification procedures used. Thus, in the case of thePPE protein, the hydrophobic nature (30% LVIFM) of the total proteincould result in its being insoluble in aqueous solvents leading to itsloss during antigen preparations. To localize the protein in thebacterial cell, high titer antibodies directed against the PPE proteinare obtained. For this, the amino-acid sequence of the peptide from gsrI-6 and gsr II-2 has been subjected to Kyte and Doolittle analyses andtwo amino acid sequences with a high antigenic index identified.

Summary of results: Sera from guinea pigs infected with airborne,virulent Mtb H37Rv, and bled within the first few weeks post-infectionhave been used to screen an expression library of Mtb DNA. Eight cloneshave been obtained by the immunoscreening. Of the 3 clones sequenced,two (gsr I-6 and gsr II-1) code for different portions of the same PPEprotein, and clone gsr II-2 codes for a protein on the RD3 region of MtbH37Rv. Thus, we have identified at-least 2 novel antigens that areexpressed by the bacteria in vivo during the time when bacterialreplication and dissemination is known to occur in this animal model.Preliminary studies suggest that sera from human TB patients haveantibodies against the fusion proteins expressed by a majority of theseclones.

EXAMPLE II Identification of Mtb Antigens that are Expressed in vivoDuring Early Stages of the Disease

In guinea pigs infected by aerosolized virulent Mtb, the in vivoreplicating organisms express molecules which are recognized by thehumoral immune system of the animals, resulting in antibody production.These antibodies, present in the sera of aerosol infected guinea pigscan be used to identify and obtain the antigens from the expressionlibrary.

The approach was to obtain antigens expressed during the early stages ofdisease progression, sera from Mtb infected guinea pigs, obtained priorto the development of clinical disease. These sera are expectf provideinformation on the GC content, presence of leader peptides (peptideswith high content of hydrophobic amino acids that are often associatedwith secreted proteins), organization of the genetic loci, etc and willenable the identification of at least some of the genes being expressed.

EXAMPLE III Confirmation that Antigens Identified in Example II areSpecific to Mtb, or Mtb Complex, and are Widely Present in ClinicalIsolates

The antigens identified by the immunoscreening may be specific to Mtb,to all members of the Mtb complex, to mycobacteria, or may be productsof genes conserved in prokaryotes.

Mtb possesses genes encoding proteins involved in house-keepingfimctions like general metabolism, signal transduction, enzymes, heatshock proteins etc that are conserved in prokaryotes. Mtb will also havegenes encoding proteins that are also present in other mycobacteria, forexample those involved in novel biosynthetic pathways that generatemycobacterial cell-wall components like mycolic acids (Ag 85 complex),lipoarabinomannans, mycocerosic acid etc. In addition the presence ofgenes encoding proteins specific to Mtb is likely. The sequencing of theMtb H37Rv genome has revealed that of the ˜4000 open reading framespresent, 20% of the encoded proteins resemble no other known proteins(5). Such specific antigens are likely to be important for diagnosticassays.

Recent studies have reported that there can be genetic differencesbetween clinical isolates of Mtb. Thus, the gene for mtp40 protein ofMtb was recently reported to be present in only some of the clinicalisolates tested (42). Similarly, the gene encoding the antigen expressedby the clone gsr II-1 in the studies presented in the progress reporthas been reported to be present in only 16% of the clinical isolates ofMtb tested (24). Only antigens that are specific to Mtb or Mtb complex,yet are widely present in clinical isolates, are likely to be useful fordiagnostic or vaccination purposes. It is therefore important to confirmthat the gene for any immunogenic protein identified in EXAMPLE II is a)specific to Mtb or Mtb complex, and b) conserved in Mtb isolates fromdifferent geographical isolates. These studies are done by 2 approaches:

a) Genomic DNA from members of the Mtb complex, clinical isolates of Mtbfrom different geographical locations, and other mycobacterial speciesare probed with genes identified in EXAMPLE II.

Materials and Methods: Clinical isolates of Mtb from 25 patients fromthe V.A. Medical Center, Manhattan, 10 patients from the Lala Ram SarupTB Hospital, New Delhi, India, and 15 patients from the Laboratoire DeSante Hygiene Mobile, Yaounde, Cameroon, Africa have been obtained.Efforts to obtain clinical isolates from patients in additionalcountries are on going. Thus, isolates from several differentgeographical regions are used in our studies. Other mycobacterialspecies (M. smegmatis, M. gordonae, M. chelonie, M. bovis BCG, M.xenopi, M. kansasi, M. fortuitum, M. africanum, M. microti etc.) havebeen obtained from the ATCC. 15-20 patient isolates of MAIS are obtainedfrom the mycobacteriology laboratory at the VAMC, Manhattan. Nonmycobacterial prokaryotes (E. coli, staphylococcus, streptococci,nocardia etc) are obtained either from the clinical microbiologylaboratory at the VAMC, or from the ATCC. Genomic DNA fromnon-mycobacterial species are isolated by routine methods (35). Forisolation of genomic DNA from mycobacterial species, standardized,published methods are used (3). Mycobacteria grown in Middlebrook 7H9broth are harvested from cultures (2-3 days old for fast growers, 14-21days for slow growers) and the pellet frozen overnight at −20° C., thenthawed and suspended in TE buffer. An equal volume ofchloroform/methanol (2:1) are added to the bacterial pellet for 5 min.to remove the cell wall lipids. The suspension is centrifuged, and thebacteria at the organic-aqueous interphase collected. These bacteria arethen suspended in TE buffer, followed by 1M Tris-HCl to raise the pHbefore addition of lysozyme and incubation overnight. This is followedby addition of an appropriate amount of 10% SDS and proteinase K to thecell lysate. The proteins are extracted by Phenol/chloroform/Isoamylalcohol extraction, the phenol removed by chloroform/isoamyl alcoholextraction, and the DNA precipitated by use of sodium acetate andIsopropanol.

Hybridization and detection methods as per the ‘DIG System User's Guidefor Filter Hybridization’ (Boehringer Manheim) are currently in use inthe lab. The genomic DNA is digested with an appropriate restrictionenzyme to completion and electrophoresed on agarose gels. The separatedDNA fragments are transferred to Hybond-N positively charged membrane(Amersham) and the DNA crosslinked to the membrane by U.V. Specificfragments are obtained from the sequence of the relevant genes (from theMtb genomic database) from genomic DNA of H37Rv by PCR, labeled with DIGand used to probe the blots prepared from the genomic DNA. Hybridizationand washing conditions are optimized for each probe, and thechemiluminescent substrate CSPD used to detect hybridization.

b) Antibodies raised against the mycobacterial peptides from antigensidentified in EXAMPLE II are used to probe lysates of the same strainsof bacteria by western blotting since it is possible that although thehomology at the DNA level is not strong, the expressed proteins may becross-reactive.

To confirm that further studies are performed only with proteins thatare specific to Mtb (or M. tb complex), and are conserved amongstclinical isolates of Mtb, the amino-acid sequence of the mycobacterialfragment in the β-gal fusion protein(s) are analyzed for regions thathave high antigenicity. A mixture of chemically synthesized peptidesrepresenting 2-4 epitopes on each fusion protein are used obtainanti-peptide antibodies from rabbits (commercially). Anti-peptideantibodies are purified from the polyclonal rabbit serum by affinitypurification and the antibodies tested to ensure that they recognize theparent fusion protein. Lysates of bacterial strains that were includedin the DNA hybridization studies are fractionated by SDS-PAGelectrophoresis and western blots probed with the anti-peptideantibodies. These experiments enable us to confirm that there are nocross-reactive proteins in the other bacterial species.

These studies will enable us to identify the antigens of Mtb or Mtbcomplex, that are conserved in clinical isolates from different sources.As mentioned in the progress report, the methods involved in thecompletion of this aim are already being used in the lab and no problemsare expected. The inclusion of M. smegnatis in the studies in EXAMPLEIII is crucial because we intend to use this as the host for expressionof the full genes of proteins of interest (see EXAMPLE 3)

EXAMPLE IV Expression in a Mycobacterial Host of the Antigens Identifiedin the Screening of Examples II & III

Expression cloning of the complete genes will provide the proteins thatcan be used for immunological studies.

The recombinant clones obtained in EXAMPLE II all express β-gal fusionproteins (FIG. 2 and 3) which contain only a fragment of the originalmycobacterial protein. For immunological studies, complete genes ofthese proteins will have to be expressed. There is increasing evidencethat proteins of Mtb expressed in the E. coli host may show differencesfrom the native counterparts. Thus, Mtb super-oxide dismutase expressedin E. coli was enzymatically inactive, whereas the same moleculeexpressed in M. smegnatis was enzymatically active (12). Reducedreactivity of human antibodies with recombinant 38 kDa protein, and 10and 16 kDa proteins has been observed (41). We compared the reactivityof sera from the same TB patients with native Ag 85C and MPT 32 purifiedfrom culture filtrates of Mtb, and with the corresponding recombinantmolecules expressed in the E. coli host (FIG. 11). Our studies showedthat human antibodies to MPT 32 and Ag 85C, that were elicited by nativeantigens during natural disease show lower reactivity with the sameproteins expressed in E. coli host (FIG. 11). Recent studies have shownthat deglycosylation of MPT 32 decreases its capacity to elicit in vitroor in vivo cellular immune responses (32). That glycosylation has a rolein proteolytic cleavage of proteins has also been shown for the 19 kDaantigen of Mtb, (17). Also, rMBP 64, expressed in E. coli was unable toelicit DTH in sensitized animals whereas the same protein expressed inM. smegmatis mimicked the native protein (31). The reasons underlyingthe differences in the immunological reactivity of native Mtb, and E.coli expressed recombinant molecules are not understood, but experiencefrom several labs shows that mycobacterium proteins expressed in amycobacterial host are immunologically more competent, probably becauseproteins expressed in E. coli lack the post-translational modificationsoften present on native Mtb antigens (15). Since vectors for efficientexpression in M. smegmatis or M. vaccae have now been constructed andused successfully (11, 12, 15), and since the recombinant proteinsobtained in EXAMPLE II are to be used for immunological studies withhuman sera, the antigens identified by the screening of the library areexpressed in mycobacterial hosts to enhance the probability of obtainingproteins that are immunologically similar to the native antigens. Sincewe intend to express only those antigens that are specific to Mtb, thestudies in EXAMPLE III will ensure that M. smegmatis does not havecross-reactive proteins or genes, the use of this organism as a hostwill not be a problem.

Materials and Methods: Two vectors that have been used successfully toexpress Mtb proteins successfully in M. smegmatis have been obtained.Vector pVV16 has been obtained from Dr. John Belisle, CSU. This vectorhas the origin of replication from pAL5000, the hygromycin resistancegene, the hsp60 promoter, and, in addition, has 6 His-tag sequences atthe C terminal end of the expressed protein. The 88 kDa protein,identified in our lab to be a potential candidate for serodiagnosis ofTB has successfully been cloned into pVV16 and expressed in M. smegmatis(FIG. 12 A). The advantage with this vector is that the hsp60 promoteris a strong promoter resulting in high level constitutive expression ofthe cloned gene. Also, the His-tag allows the use of commerciallyavailable Nickel-Agarose columns (Qiagen) for purification of the clonedprotein. The basic method for cloning specific genes into any expressionvector is described (11). Briefly, PCR amplification of the target geneare performed using primers that contain restriction sites to generatein-frame fusions. The PCR product are purified and digested with theappropriate restriction enzymes and purified again. The vector DNA willalso be cut with the appropriate restriction enzymes and purified. ThePCR product and the vector are ligated, electroporated into DH5 andplated onto hygromycin containing plates overnight. Several antibioticresistant colonies are grown in small volumes of medium, and the plasmidDNA isolated by miniprep. The size of the insert is checked in thesecolonies. Inserts from one or more colonies are sequenced to ensurefidelity of the amplified gene.

For electroporation into M. smegmatis, the bacteria are grown shaking in7H9 medium till an OD of 0.8-1.0 is obtained. The bacteria areharvested, washed twice with water, once with 10% glycerol and suspendedin the same. An aliquot of the M. smegmatis cells is electroporated withthe plasmid DNA from the colony whose insert was sequenced. Theelectroporated cells are grown for 34 hrs in 7H9, and plated onantibiotic containing plates. Several resistant colonies are grown inminimal media for 48-72 hrs. The bacterial cells are collected, frozenin liquid nitrogen overnight, thawed, suspended in PBS containingprotease inhibitors, and sonicated in ice for 5 mins. After centrifugingthe lysate for 30 mins at 5000 rpm, the supernatants are aliquoted andfrozen. Five-10 ul of the lysate is fractionated on 10% SDS-PA gels, andwestern blots prepared from these gels are probed with anti-Hisantibodies to confirm the expression of the protein.

The protein(s) are purified from the lysates by use of commercialNickel-chelate-nitrilotriacetic acid (Ni-NTA) columns (Quiagen, Inc)(19). These columns allow the purification of proteins constituting <1%of the total cellular protein to >95% homogeneity in one step. Briefly,the M. smegmatis containing the cloned genes are grown in Middlebrook7H9 medium for 72 hrs, after which the bacteria are pelleted bycentrifugation and resuspended (1/100 volume) in PBS containing proteaseinhibitors (PMSF, DTT, EDTA). The bacterial cell pellet is frozenovernight in liquid nitrogen overnight, thawed and exposed to 1 mg/mllysozyme for 30 mins (in ice). The pellet will then be sonicated and thelysate treated with RNase and DNase for 30 mins. The lysate will then besubjected to high speed centrifugation (>10, 000 g for 20 mins) and thesupernatant mixed with an equal volume of slurry of Ni-NTA agarose inthe appropriate buffer. The His-tagged protein is allowed to bind to theagarose for 60-90 mins in ice, the mix is loaded onto a column, andwashed 2-3 times with buffer to get rid of unbound material. The boundprotein will then be eluted by use of appropriate elution buffer. Thepurification procedures for His-tagged proteins may need to be modifiedfor different proteins (19, 20), and the specific conditions for eachprotein is developed and optimized in consultation with Dr. JohnBelisle.

As mentioned above, the 88 kDa protein of Mtb has been successfullycloned into this vector (FIG. 12A). Lysates of M. smegmatis mc2 aloneand mc2 with the 88 kDa-pVV16 (10 μg/lane) were fractionated by SDS-PAGEand western blots probed with anti-His antibodies. The His-tagged 88 kDarecombinant protein is well expressed and easily detectable.

One possible problem that may be encountered with one or more of theproteins cloned in this vector is that accumulation of foreign proteinscan sometimes lead to toxicity to the host cell, or the recombinantprotein forms inclusion bodies which necessitates denaturation of theprotein for purification. An alternative vector, pDE 22 has beenobtained from Dr. Douglas Young, imperial college, London. This vectoris derived from a vector pSMT3 which has been used successfully forexpression of 4 different Mtb proteins (11, 15), and also contains thepAL5000 origin of replication, the gene for hygromycin resistance, thehsp60 promoter and has the signal sequence from BCG alpha gene. In thiscase, the recombinant protein is secreted out of the host, and sotoxicity to the host or inclusion body formation is not a problem.Moreover, this vector can also be used for expression in M. vaccae ifrequired. The proteins cloned into pDE 22 are secreted out of the host,and the recombinant protein is present in the culture supernatants. Oneproblem that may be encountered in the use of this plasmid is that theM. smegmatis host itself may express proteins that cross-react with theMtb protein. To determine if cross-reacting extracellular antigens arepresent in culture filtrates of M. smegmatis, the organisms were grownin minimal media. The culture supernatants obtained after 24, 48 and 72hrs of growth were concentrated 30-fold by Amicon filtration (10 kDacut-off), 10 μgs fractionated on a 10% SDS-PA gel and 2 identical blotscontaining fractionated, concentrated culture filtrate proteins andLAM-free CFP (as positive control) probed with TB sera or healthycontrol sera. The TB sera recognized several proteins in the LAM-freeCFP preparation, but no specific bands in the concentrated M. smegmatisculture filtrate (FIG. 12B). The healthy control sera showed noreactivity with either of the antigen preparations. These results showthat M. smegmatis itself does not produce any extracellular proteinsthat cross-react with sera from TB patients.

EXAMPLE V Assessing the Role of the Recombinant Proteins in HumoralResponses

Antibodies to the recombinant antigens identified in EXAMPLES II and IIIare present in the sera of individuals with clinical and/or subclinicalactive TB.

Scant information is available on the Mtb antigens that are expressed bythe in vivo bacteria and are recognized by the human immune systemduring the early stages of disease progression. The guinea-pig sera usedto obtain the antigens was from animals that had been infected withaerosolized, virulent, Mtb and bled before they developed the disease.The antibodies in the sera of the animals at this stage are directedagainst antigens that are expressed by the bacteria during thispre-clinical period of bacterial replication and dissemination. Theprofile of antigens of Mtb recognized by tuberculous guinea pigs andhumans is very similar (FIG. 7). Moreover, human TB serum pools showreactivity with fusion proteins from several clones (FIG. 8). The fusionprotein encoded by clone gsr 1-6 is recognized by sera from TB patientsbut not by sera from healthy controls (FIG. 9). The HIV-pre TB serumpool recognizes at least 2 of the antigens (FIG. 10). Together theseresults support the hypothesis that the antigens identified by guineapig sera will also be recognized by the human TB sera. Evaluation ofreactivity of proteins identified in this study with sera from patientsat different stages of disease will enable us to determine which ofthese antigens is recognized by humans during early TB.

Materials and methods: In order to determine the stage of TB infectionat which antibodies to these antigens are present in humans, and theirutility in serving as markers of active infection, antibodies to themare assessed in serum samples in the already existing cohorts in thelab. Sera from the following cohorts are available in the PI'slaboratory.

Sera from non-TB controls: Sera from 40 PPD positive and 60 PPD negativehealthy controls are available in the laboratory. These sera are used asnegative controls for assessment of antibodies to the recombinantantigens in the sera from TB patients at different stages of diseaseprogression. Sera from ˜50 HIV-infected, asymptomatic individuals arealso available, and are included as additional controls.

Pre-TB and TB sera from HIV-infected individuals: We currently have serafrom >50 HIV-infected patients who developed clinical TB {and >200 serumspecimens from the same subjects that were obtainedprior to theirdeveloping TB (pre-clinical TB)}. These are HIV-infected individuals whowere being regularly monitored for their T cell profiles, and developedclinical TB during the course of the HIV disease progression.Serum/plasma samples from each time when the T cell profiles wereevaluated was saved, providing us with retrospective sera that wereobtained prior to clinical manifestations of TB, that is, duringpre-clinical disease (21). Chest X-ray reports, and microbiological datafrom these patients are also available]. This is the earliest stage ofactive infection that can be recognized in humans. Since these sera arefrom pre-clinical stages of tuberculosis, they should contain antibodiesto the antigens expressed during early stages of tuberculosis diseaseprogression. This is a unique, well-characterized and extremely valuableset of specimens which, to the best of our knowledge, does not existanywhere else. Only with such a specimen bank could these studies beundertaken

Sera from minimal TB patients: Serum samples from 20 patients withnon-cavitary TB are available in the laboratory. A majority of thesepatients are also smear negative for Acid Fast Bacilli. These patientsare at a relatively early stage of disease progression, defined as“early TB” or “early infection.” These patients are from the ManhattanVA Medical Center. Sera from additional patients with a similar clinicalprofile are obtained with informed consent, during the course of thestudies.

Sera from advanced TB patients: Serum samples from 60 cavitary, smearpositive patients have been obtained from India and from about 20similar patients from the Manhattan VA medical center. These serarepresent samples obtained at an advanced stage of disease progression.

Sera from HIV-infected individuals with M. avium bacteremia: The pre-TBsera are derived from HIV-infected patients. These patients are also athigh risk for having M. avium infection. To further ensure thespecificity of the antibody responses to the recombinant antigens, serafrom 20 HIV-infected individuals who developed M. avium bacteremia,obtained at the time of disease manifestation, and in the months oryears prior to the bacteremia (equivalent to the pre-TB and at-TB serafrom HIV-TB patients) have also been obtained. These sera will also beused as negative controls.

The sera in the above cohorts represent sera obtained at differentstages of disease progression in humans. Reactivity of these sera withthe purified proteins are assessed by ELISA. The method used is the sameas described in our previous publications. Briefly, the recombinantantigens purified from M. smegmatis supernatants or lysates are used tocoat the ELISA plates at a predetermined optimal antigen concentrationovernight. Next morning, the plates are washed, blocked with PBScontaining 5%BSA and 2.5% FCS for 2.5 hrs. This is followed by theaddition of a predetermined optimal dilution(s) of the serum samples tothe antigen-coated wells. After incubating the antigen coated wells withantibody containing sera for 90 mins, the plates are washed with PBScontaining 0.05% Triton-X and then exposed to alkalinephosphatase-conjugated anti-human IgG, followed by the substrate for theenzyme. We routinely use the GIBCO-BRL amplification system as thesubstrate since it increases the sensitivity of antibody detection.Checker-board titration is used to determine the optimal antigenconcentration, and serum dilution for each antigen. For MPT 32 and Ag85C, as little as 50 μls per well of a 2 μg/ml suspension of thepurified protein was required for optimal results. Mean optical densityplus 2.5-3 SD with the sera from the healthy individuals is used as thecut-off to determine positive reactivity of patients. Reactivity withthe guinea pig sera which were used for the initial immunoscreening ofthe λgt11 library is included as positive control.

Studies with the above cohorts of sera will help to identify theantigens that are expressed by the in vivo M. tb, and recognized by theimmune system during different stages of disease progression in humans.We expect that some antigens (that are expressed by the in vivo bacteriaat all stages of disease progression) are reactive with antibodies frompatients at all different stages of TB described in the cohorts above.Antibodies to these antigens are absent in various groups of negativecontrols (including the PPD+ healthy individuals). Such antigens arevery useful for devising diagnostic tests since a single test could thenbe used to diagnose TB at any stage of the disease progression. However,the in vivo bacteria may express some antigens only during early stagesof TB, and not during advanced TB. Such antigens would be recognizedonly by antibodies obtained during early TB, for example by the pre-TBsera from the HIV-infected individuals. Such antigens are useful fordevising tests for identification of individuals who are at high risk ofdeveloping infectious TB.

Reactivity of sera from a cohort of individuals at high risk ofdeveloping TB: Dr. J. J. Ellner, TB Research Unit (TBRU), Case WesternReserve University, had initiated studies with a cohort comprising offamilies with one or more index cases of confirmed TB during the last 2years in Uganda. Three hundred and two families with at least one smearpositive TB case are included in the study, with approximately 1200household contacts. All contacts were evaluated for clinical TB, TBinfection and underlying diseases that may predispose to TB at the timeof inclusion into the study.

Over the past year, 14 of the household contacts who did not initiallyhave any signs and symptoms of TB have developed TB during follow up.Baseline sera (obtained at the time of inclusion into the study), andsera obtained during follow up from contacts who developed TB during thecourse of the study are evaluated for reactivity with the antigensobtained in EXAMPLE IV. An equal number of household contacts who didnot develop TB, and are members of the same families are included inthis testing as negative controls. Any additional contacts who developTB during the course of the study will also be included in thesestudies. This longitudinal study is designed to determine which antigenscan be used as surrogate markers for identification of individuals whoare at a risk of developing TB in high-risk populations. The selectionof the individuals and the sera, the number and appropriateness of thecontrols and the analysis of the data is done in consultation with Dr.Christopher Whalen, Epidemiology leader for the TB Research Unit.

Cohort of recent converters of PPD reactivity: The VA assesses the PPDskin test reactivity of all employees and volunteers (1500 individuals)on an annual basis. Of these, ˜500 are baseline positive. Allindividuals working in the emergency room, medical intensive care unitand those involved in taling care of the TB patients are tested everysix months. About 5-10 individuals convert to positive reactivity everyyear. The testing is done with 5 US units/test, of tuberculin obtainedfrom Pasteur-Meriuex Corporation, and 10 mm or greater induration isconsidered positive. Sera from recent converters of PPD skin test areobtained with their informed consent. The reactivity of the seraobtained from recent converters with the recombinant antigens isdetermined by the above described methods, and compared to thereactivity with an equal number of long term PPD positive individuals.Since these individuals are employees of the hospital, both male andfemale individuals are included in the cohort.

Summary: This study focuses on identifying, obtaining and studying theantigens of Mtb which are expressed by the bacterium during in vivoreplication. No such antigens that are associated with early TB havebeen described before. The fact that one of the antigens we identifiedis a PPE protein is interesting, since other pathogens have similarproteins, which elicit cellular and humoral immune responses in theirhosts, and also contribute to immune evasion by antigenic variation

The studies of humoral responses elicited by these antigens contributeto the development of diagnostic assays. If, as in guinea-pigs, humansalso recognize one or more of these proteins prior to clinicalmanifestation of TB, these antigens can be included in tests that can beused to screen large numbers of suspect individuals quickly. Thedetection of individuals with early, subclinical disease will enableclinicians to institute treatment to patients before they developdisease and become infectious. This will benefit not only theindividuals themselves, but also contribute significantly to decreasingthe transmission of the infection in the community.

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34 Sada, E., L. E. Ferguson, and T. M. Daniel. 1990. An ELISA for theserodiagnosis of tuberculosis using a 30,000-Da native antigen ofMycobacterium tuberculosis. J. Inf Dis. 162:928-931.

35 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

36 Skurnik, M., and P. Toivanen. 1993. Yersinia entercoliticalipopolysaccharide: genetics and virulence. Trends Microbiol. 1:148-152.

37 Smith, D. W., D. N. McMurray, E. H. Wiegeshaus, A. A. Grover, and G.E. Harding. 1970. Host parasite relationships in experimental airbornetuberculosis IV. Early events in the course of infection in vaccinatedand nonvaccinated guinea pigs. American Rev. of Respiratory Disease.102:937-949.

38 Smith, D. W., and E. H. Wiegenshaus. 1989. What Animal Models CanTeach Us About the Pathogenesis of Tuberculosis in Humans. Reviews ofInfect. Dis. 2:s385-s393.

39 Sonnenberg, M. G., and J. T. Belisle. 1997. Definition ofMycobacterium tuberculosis culture filtrate proteins by two-dimensionalpolyacrylarnide gel electrophoresis, N-terminal amino acid sequencingand electrospray mass spectrometry. Infect. Immun. 65:4515-4524.

40 Triccas, J. A., F.-X. Berthet, V. Pelicic, and B. Gicquel. 199. Useof fluorescence induction and sucrose counterselection to identifyMycobacterium tuberculosis genes expressed within host cells.Microbiology. 145:2923-2930.

41 Verbon, A. 1994. Development of a serological test for tuberculosis.Trop Geo Med. 46:275-279.

42 Weil, A., B. Pikaytis, W. Butler, C. Woodley, and T. Shinnick. 1996.The mtp40 gene is not present in all strains of Mycobacteriumtuberculosis. J. Clin. Microbiol.:2309-2311.

43 Wiegeshaus, E. H., D. N. McMurray, A. A. Grover, G. E. Harding, andD. W. Smith. 1970. Host-parasite relationships in experimental airbornetuberculosis III. Revelance of microbial enumeration to acquiredresistance in guinea pigs. Am. Rev. Respir. Dis. 102:422-429.

44 Young, D. B., S. H. E. Kaufinann, P. W. M. Hermans, and J. E. R.Thole. 1992. Mycobacterial protein antigens: a compilation. Mol.Microbiol. 6:133-145.

45 Young, R. A., B. R. Bloom, C. M. Grosskinsky, J. Ivannyi, D. Thomas,and R. W. Davis. 1985. Dissection of Mycobacterium tuberculosis antigensusing recombinant DNA. Proc. Natl. Acad. Sci. USA. 82:2583-2587.

EAMPLES VI-XIII

Parts of the studies described in Examples VI-XIII below were alsopublished in K. K. Singh, X. Zhang, A. Sai Patibandla, P. Chien and S.Laal: Antigens of Mtb Expressed During Pre-Clinical TB: SerologicalImmunodominance of Proteins with Repetitive Amino Acid Sequences, Infec.Immun. 69:4185-4191 (1991), which is incorporated by reference in itsentirety. References cited in these Examples as numbers in parenthesesare listed following Example XIII.

EXAMPLE VI Materials and Methods

Serum samples from rabbits: Six pathogen-free rabbits (2.5 to 2.7 kg,Covance Research Products, Inc., Denver, Pa.) were infected by aerosolsof Mtb H37Rv and another six were similarly infected by aerosol of MtbCDC 1551 at the US Army Medical Research Institute of infectiousDiseases, F. Detrick, Frederick, Md. (10). After infection, the rabbitswere maintained in the BL3 facility at George Washington University,Washington, DC. The infected rabbits were bled 5 weeks post-infectionwhen they were euthanized for determination of tubercles in their lungs(6). All 12 rabbits showed the presence of tubercles, confirming thatall animals had been successfully infected. Also, there was nosignificant difference in the numbers of tubercles, or in the bacterialloads in the tubercles, between the rabbits infected by the H37Rv orCDC1551 strains(6). Sera from 3 normal (uninfected) rabbits was obtainedas controls.

Serum samples from Humans: Sera were obtained from the following groupsof individuals:

-   a) 5 PPD sldn test positive, healthy individuals. Three of the 5    individuals were BCG vaccinated, and all 5 would also be potentially    exposed to the bacteria since they were individuals working in the    laboratory or were clinicians working in the VA Infectious Disease    Clinic.-   b) 10 HIV-infected patients: This patient cohort has been described    earlier (24). Briefly, these were HIV-infected individuals who were    routinely being monitored for their CD4 numbers, and developed TB    during the course of HIV disease progression. At each time point    when they were bled for evaluation of the T cell numbers, plasma    from these patients had been saved and frozen. Thus, when they    developed TB, it was possible to identify and obtain the    retrospective, pre-TB sera. Multiple samples from each individual    are available, but for the current study, one randomly chosen pre-TB    serum obtained around 6 months prior to clinical TB was used for    each individual.-   c) 2 TB patients with early disease: These were smear negative,    culture positive TB patients with infiltration in their lungs but no    radiological evidence of cavitary lesions.-   d) 5 TB patients with advanced disease: These were smear positive TB    patients with extensive cavitary lesions.

Mtb H37Rv antigen preparations : Two antigen preparations of Mtb H37Rv,lipoarabinomannan (LAM)-free culture filtrate proteins (LFCFP), andSDS-soluble cell-wall proteins (SDS-CWP) were tested in the study. Thepreparation of these antigens has been described previously (25). Theformer antigen preparation contains >100 different proteins, some ofwhich (˜30%) have been mapped on the basis of reactivity with murinemonoclonal antibodies or peptide sequencing (41). The latter preparationcontains˜estimate different proteins but these have not been mapped asyet.

Immunoscreening of Mtb λgt11 library: Mtb λgt11 expression library wasobtained from the World Health Organization (47). The library containsrandom sheared fragments of Mtb H37Rv DNA cloned into gt11 phage thatexpresses the foreign insert DNA as E. coli β-galactosidase (β-gal)fusion protein. Immunoscreening of expression library was performed bystandard methods (47). Briefly, E. coli Y1090 was infected with phagefrom the library and plated in top agar on LB plates. After 2.5 hincubation at 42° C., expression of recombinant proteins was induced byoverlaying the plates with Isopropyl β-D thiogalactoside (IPTG, Sigma)saturated nitrocellulose filters for 2.5 h at 37° C. The filters wereremoved, washed and probed with 1:50 dilution of a serum pool from theabove described 12 infected rabbits. The serum pool was absorbedextensively with an E. coli lysate before use. The recombinant phagesproducing positive signals were cloned and designated as AD clones.

Western Blot analysis: This was used both for evaluating reactivity ofthe rabbit sera with the LFCFP and the SDS-CWP preparations, as well ascharacterization of the recombinant proteins expressed by the AD clones.Briefly, the Mtb antigen preparations were fractionated on 10% SDS-PAgels, and the western blots probed with serum pools from Mtb infected oruninfected rabbits. The blots were washed with PBS, and blocked with PBScontaining 3% BSA for 2 h. After washing the blots with PBS-2% Tween 20(PBST), they were incubated overnight with the rabbit pools described ata dilution of 1:60 in PBST-1% BSA at 4° C. after which they were washedwith PBST and exposed to 1:2000 dilution of alkalinephosphatase-conjugated goat anti-rabbit IgG (Sigma, St Louis, Mo.) for1.5 h. Extensively washed blots were developed with BCIP-NBT substrate(Kirkegard & Perry Labs, Gaithersburg, Md.).

For the recombinant protein studies, lysogenic strains were preparedfrom phage clones in E. coli Y1089 (37). Single colonies from lysogenswere grown in LB medium at 32° C. till midlog (optical density of 0.5 at600 nm), induced at 45° C. for 20 min and followed by addition of IPTG(10 mM) and further incubation at 37° C. for 1 h. The bacterial pelletsobtained were sonicated in a small volume of PBS containing 1 mM of DTT,EDTA and PMSF, and the lysates fractionated on 10% SDS-PAGE gels. Thewestern blots were probed as described above with the serum pool frominfected rabbits (1:200), or with a pool of sera from uninfected rabbits(1:200) or with murine anti-β-gal monoclonal antibody (1:10,000)(Promega), or with human sera (1:100-1:700) and the appropriate alkalinephosphatase-conjugated IgG secondary antibody. Lysates from E. coliY1089 lysogenized with λgt11 phage without insert were included ascontrols.

Isolation, sequencing and computer analysis of DNA from recombinantλgt11 clones: DNA from recombinant λgt11 clones was isolated by usingQiagen λ DNA purification kit (Qiagen, Valencia, Calif.), digested withEcoRI for release of mycobacterial DNA insert, and the insert DNApurified by extracting from low melting agarose gel with QIAquick gelextraction kit (Qiagen,). The purified EcoRI insert DNA was subclonedinto vector pGEMEX-1 (Promega, Madison, Wis.) at the EcoRI site and therecombinant plasmid transformed into JM 109 competent cells. Therecombinant plasmid DNA was isolated using Wizard plus minipreps kit(Promega), and used for sequencing with SP6 and T3 promoter primersflanking the multiple cloning site in pGEMEX-1. The sequence similarityanalysis of the DNA sequences was performed by BLAST using the NationalCenter for Biotechnology Information site (NCBL USA). The repetitivestructures in the protein were analyzed by using Statistical Analyses ofProtein sequences (SAPS). Prediction of trans-membrane helices wasperformed by TMpred software using ISREC server at European MolecularBiology Research network-Swiss node site. The prediction of signalpeptide and signal peptidase cleavage sites were performed by theSignalP V2.0 software using neural networks (NN) and hidden Markovmodels (HMM) trained on gram positive bacteria, available from Centerfor Biological Sequence Analyses (CBSA, Denmark). The glycosylationsites were predicted by using the NetOGlyc 2.0 software also availablefrom the CBSA. The Kyte & Doolittle hydrophobicity plot and theoreticalmolecular weight and pI of the proteins were performed by using softwarefrom ExPASy site. Prosite profile scan was performed by ISREC server atSwiss Institute for Bioinformatics (SIB).

EXAMPLE VII Reactivity of Mtb Infected Rabbit Serum Pool with LFCFP andthe SDS-CWP Preparations

Studies with other intracellular bacterial pathogens suggest that thefirst crucial steps towards establishment of the infecting organism,adhesion and invasion, are likely to be mediated by extracellularlyexpressed or cell surface associated proteins of the pathogen (21, 22,32). To determine if any antigens in the culture filtrates or cell-wallpreparations of Mtb are recognized by antibodies from the infectedrabbits at 5 weeks post-infection, a pool of sera from all 12 rabbitswas used to probe the LFCFP and SDS-CWP preparations (FIG. 13), and thereactivity compared with a pool of sera from 3 uninfected rabbits. Threebands corresponding to ˜27.5 kDa, 35.5 kDa and 56 kDa in LFCFPpreparation were reactive only with the serum pool from the infectedrabbits, as were two bands corresponding to 27.5 kDa and 43 kDa in theSDS-CWP preparation (FIG. 13, lane 3 and 5). The serum pool from theinfected rabbits was absorbed extensively against a lysate of E. coliY1090 and used to screen the λgt11 expression library of Mtb H37Rvgenomic DNA (47).

EXAMPLE VIII Screening of the λgt11 Library and Characterization of theRecombinant Proteins

To obtain the antigenic proteins that are recognized by antibodies inthe sera from the Mtb infected rabbits, ˜1.2×10⁵ pfu from the librarywere screened with the serum pool. Seven clones were plaque purified,and designated AD1, AD2, AD4, AD7, AD9, AD10, and AD16.

Lysates prepared from cultures of single colonies of lysogens of all 7AD clones were fractionated on 10% SDS-PAGE, and the western blotsprobed with the rabbit serum pools from infected or uninfected rabbits,or mouse anti-β-gal monoclonal antibody. As shown in FIG. 14, all 7recombinant clones produced β-gal fusion proteins, with sizes rangingfrom 125 kDa to 170 kDa, which were recognized both the anti-β-gal mAb(FIG. 14 lanes 2-8) and with the rabbit serum pool from the infectedrabbits (FIG. 14, lanes 20-26). The recombinant fusion proteins failedto react with the serum pool prepared from uninfected rabbits (FIG. 14,lanes 11-17).

DNA Sequence and Protein Analyses

Restriction digestion 5 of the 7 clones with EcoRI yielded single insertranging from 3.7 kb to 5.6 kb. The remaining clones had multipleinserts. This manuscript reports the results obtained with the fiveclones with single EcoR1 inserts. Sequencing of the EcoRl inserts of the5 clones after subcloning into PGEMEX-1 resulted in sequencing of about450-700 bp nucleotides from each end. Orientation of the insert in theAD clones were determined by restriction map analysis (data not shown).

EXAMPLE IX Sequence Analyses of Clones AD1 and AD2

DNA sequence analyses of both ends of EcoR1 insert of clones AD1 (5.1kb) and AD2 (4.6 kb) showed 98% identities to different regions of twooverlapping cosmids MTV026 and MTCY409. One end of the insert of cloneAD1 (nu 1-440) showed homology to nu 23458-23740 of cosmid MTV026 and nu1-207 of overlapping cosmid MTCY409 while the other end (nu 4530-5154)showed homology to nu 4297-4921 of cosmid MTCY409 (FIG. 15A). Similarly,one end of the insert of clone AD2 (nu 1-610) showed homology to nu23227-23740 of cosmid MTV026 and nu 1-146 of cosmid MTCY409 while theother end (nu 3958-4620) showed homology to nu 3494-4156 of cosmid MTCY409 (FIG. 15A). Restriction map analysis showed that the ends of theinserts of clones AD1 and AD2 which showed homology with cosmid MTV026was in correct reading frame with β-gal.

The peptide expressed in clones AD1 (nu 1-123) and AD-2 (nu 1-354)represents amino acids 245-284 and 168-284 respectively in theC-terminal region of Rv3810 (pirG) gene product. The protein encoded bythe Rv3810 (pirG) is a 284 amino acid cell surface protein precursor,which is almost identical (99.3% identity in 284 aa overlap) topreviously described cell surface protein ERP (exported repetitiveprotein) of Mtb (4) and secreted antigen p36/p34 (5) of M. bovis. Thisgene also shows 53.4% identity to a M. leprae gene for a 28 kDa protein(7). As reported earlier, the ERP protein has 12 tandem repeats of fiveamino acid PGLTS in the central region from position 92 to 173. Thetheoretical molecular weight and pI of the protein are 27.6 kDa and 4.34respectively, although the molecular weight of the native molecule wasreported to be 36 kDa (3). The protein has a typical N-terminal signalsequence with a possible signal peptidase cleavage site at position 22.The Kyte-Dolittle plot demonstrated hydrophobic regions in theN-terminal and C-terminal portion of the protein which have no repeatmotifs and a hydrophilic central portion which contains all the repeatmotifs.

EXAMPLE X Sequence Analyses of Clone AD9

DNA sequence analyses of both ends of EcoR1 insert of clone AD9 (4.9 kb)showed 94% identities to different regions of cosmid MTV004. One end ofthe insert (nu 1-540) showed homology to nu 36201-36740 and other end(nu 4364-4921) to nu 40564-41121of the cosmid (FIG. 15B). Restrictionmap analysis showed that the end of the insert of clone AD9 which is incorrect reading frame with β-gal, starts within the gene Rv 3367(PE-PGRS). The peptide expressed in clone AD9 (nu 1-1080) representsamino acids 230-588 in the C-terminal region of Rv3367 (PE-PGRS) geneproduct (FIG. 16).

The protein encoded by the Rv3367 (PE_PGRS) is a 588 amino acid protein,which is a member of recently described PE-PGRS family of glycine-richMtb proteins (9). This protein possesses the highly conserved N-terminaldomain of ˜110 residues and Pro-Glu (PE) motif near the N-terminusdescribed to be characteristic of the PE protein family (9). The geneproduct of Rv3367 showed the presence of 39 tandem copies of motifGly-Gly-Ala/Asn and 43 tandem copies of motif Gly-Gly-X (total 82repeats) spanning the entire protein except the conserved N-terminalregion (FIG. 16). The deduced amino acid sequence encoded by clone AD9contains 61 repeats of the motifs. Amino acid analysis of Rv3367 by SAPSpredicted five other possible repetitive motifs,Gly-Asn-Gly-Gly-Asn-Gly-Gly, Gly-Asn-Gly-Gly-Ala-Gly-Gly,Asn-Gly-Gly-Ala-Gly-Gla-Asn,Gly-Gly-Ala-Gly-Gly-Ala andGly-Ala-Gly-Gly-Asn-Gly-Gly in the region extending from aa 137-542. Thetheoretical molecular weight and pI of the protein are 49.7 kDa and 4.05respectively. This protein has a high content of Gly (38.32%), Ala(16.26%) and Asn (8.97%). The homology search showed 50-55% homology tomost of the members of PE-PGRS family of Mycobacterium tuberculosisH37Rv. This protein also displayed homology with a glycine richcell-wall structural protein of Phasiolus Vulgaris (42% identity in 483aa overlap). The Kyte-Dolittle plot demonstrated a hydrophobic region inN-terminal portion of the protein with no repeat motif clusters, and ahydrophilic C-terminal which has the majority of the repeat motifs. AN-terminal signal peptide with a putative signal peptidase cleavage sitebetween aa 44 and 45, and two putative O-glycosylation sites atpositions 221 and 438 are predicted to be present in the protein. TMpredanalysis predicted five transmembrane helices at aapositions 24-43,166-186, 194-218, 351-368 and 431-451.

EXAMPLE XI Sequence Analyses of Clone AD10

DNA sequence analyses of both ends of EcoR1 insert of clone AD10 (3.7kb) showed 94% identities to different regions of cosmid MTY25D10. Oneend of the insert of clone AD10 (nu 1-623) showed homology to nu17037-17659 and other end (nu 3147-3742) to nu 20183-20778 of cosmidMTY25D10 (FIG. 15C). Restriction map analysis showed that the end of theinsert of clone AD10 which is in correct reading frame with β-gal,starts within the gene Rv0538. The peptide expressed in clone AD10 (nu1-636) represents amino acids 338-548 in the C-terninal region of Rv0538gene product (FIG. 17). The protein encoded by Rv0538 is a 548 aminoacid hypothetical protein with a repetitive proline and threonine-richregion at C-terminal (proline threonine repetitive protein, PTRP). Aminoacid analysis of Rv0538 (PTRP) gene product showed the presence of 23tandem repeats of motif Pro-Pro-Thr-Thr in C-terminal region fromposition 415 to 495, with positions 2, 3 and 4 being better conserved ascompared to position 1. The deduced amino acid sequences encoded byclone AD10 contains all 23 repeats of the motif (FIG. 17). SAPS aminoacid analysis of Rv 0538 (PTRP) gene revealed 7 tandem repeats of motifThr-Thr-Pro-Pro-Thr-Thr-Pro-Pro-Thr-Thr-Pro-Val from aa 413 to 489. Thetheoretical molecular weight and pI of the protein are 55 kDa and 4.44respectively.

This protein has a high content of Proline (15.63%), Alanine (15.23%),Threonine (12.83%) and valine (11.42%) with two proline rich regions ataa positions 334-340 and 387-464. No signal peptide appears to bepresent but four transmembrane helices at aa positions 97-114, 198-218,278-299 and 379-398 and 50 putative O-glycosylation sites, mostly atC-terminal, are predicted. The Kyte and Doolittle plot shows thepresence of seven short hydrophobic regions in the protein. Homologysearches showed 100% identity in C-terminal region to a 295 aa (29.4 kD)hypothetical Mycobacterium bovis protein and 40% identity in 226 aaoverlap to a probable cell wall-plasma membrane linker protein ofBrassica napus.

EXAMPLE XII Sequence Analyses of Clone AD16

DNA sequence analyses of both ends of EcoR1 insert of clone AD16 (5.6kb) showed 98% identities to different regions of cosmid MTY20B11. Oneend of the insert of clone AD16 (nu 1-628) showed homology to nu24404-23777 and other end (nu5028-5646) showed homology to nu19377-18759 of cosmid MTY20B11 (FIG. 15D). Restriction map analysisshowed that the end of the insert of clone AD16 which is in correctreading frame with β-gal, starts within the gene Rv3246c (mtrA). Thepeptide expressed in clone AD16 (nu 1-216) represents amino acids157-228 in the C-terminal region of Rv3246c (mtrA) gene product. Theprotein encoded by the Rv 3246c is a 228 amino acid MtrA responseregulator protein, a putative transcriptional activator, which isidentical (100% identity in 225 aa overlap) to previously describedresponse regulator protein MtrA of a putative two-component system,mtrA-mtrB of Mtb H37Rv (44) and similar (55.2% identity in 221aaoverlap) to M. bovis regX3 [#1838). A homolog of the Mtb MtrA proteinwas also identified in cell wall fraction of M. leprae (30). Thetheoretical molecular weight and pI of the protein are 25.2 kDa and 5.34respectively. The Kyte and Doolittle plot showed presence of hydrophobicregion in the N-terminal of the protein.

EXAMPLE XIII Reactivity of Recombinant Proteins with Sera fromIndividuals with TB at Different Stages of Disease Progression

In order to determine if the antigens identified by sera from aerosolinfected rabbits were expressed during human infection with Mtb, theirreactivity with sera from TB patients was evaluated. Initially pooledsera from individuals at different stages of disease progression wereused. The fusion proteins of PE-PGRS protein (FIG. 18A), the PTRP (FIG.18B) and the MtrA (FIG. 18C) were strongly reactive with pooled serafrom the pre-TB patients. The PE-PGRS protein was also well recognizedby the serum pools from non cavitary and the cavitary TB patients (FIG.18A), but the PTRP and the MtrA fusion proteins showed poorer reactivitywith these serum pools (FIG. 18B and D). In contrast, the pirG (ERP)fusion protein reacted only with the serum pool from the cavitary TBpatients (FIG. 18C).

Since the pre-TB serum pools showed reactivity with fusion proteins ofthree of the four antigens, reactivity with pre-TB sera from 10individual patients and 3 PPD positive controls was assessed. All 10pre-TB sera recognized the PE-PGRS (FIG. 19A) and PTRP (FIG. 19B) fusionproteins, whereas 6 of the 10 patients had antibodies to the MtrA fusionprotein (FIG. 19C). None of 10 patients showed reactivity with the pirG(ERP) fusion protein when tested individually (data not shown).

DISCUSSION OF EXAMPLES VI-XIII

The rabbit model of TB closely resembles TB in immuno-competent humansin that both species are outbred, both are relatively resistant to Mtb,and in both the infection may or may not progress to form liquified fociand cavities(6). The paucity of human material available for study ofimmunological events occurring after inhalation of virulent bacillinecessitates the use of animal models for these studies. The sera usedin this study was obtained from rabbits at 5 weeks post-infectionbecause earlier studies have shown that the logarithmic multiplicationof inhaled Mtb within the lungs of infected rabbits slows down at about3 weeks post-infection, and the 4^(th) week onwards, the numbers ofcultiviable bacilli decrease (11). Thus, immune responses that caninhibit the intracellular multiplication of inhaled Mtb are firstrecognized at 4-5 weeks post-infection. Using antibodies in these seraas markers of antigens expressed in vivo, 4 antigens from the Mtbexpression library were recognized. Two of these are novel proteins, oneis a member of PE-PGRS family of proteins and the other is a proteinwith proline threonine repeats (PTRP). The other proteins identified inthis study, the pirg (ERP), and the MtrA were previously identified byother methodologies, although their role in natural infection anddisease progression has not been explored (4, 44).

Interestingly, all four proteins identified by the use of earlypost-infection sera are either known to be, or have signatures of,surface or secreted proteins of Mtb. Thus, the pirG (ERP) protein hasbeen shown to be a cell surface-exposed protein that is expressed by thebacteria during residence in the phagosomes of in vitro maintainedmacrophages (3). The cellular location of the Mtb MtrA is not known, butthe homolog of MtrA was isolated from cell walls of M. leprae (30). Thiscell surface location of the Mtb MtrA is consistent with its proposedrole as response regulator of a putative two component system mtrA-mtrB(44). The PE-PGRS protein has a hydrophobic N terminal, a putativeN-terninal signal peptide and 5 transmembrane regions, suggesting thatthe protein is either secreted or cell surface associated. Theprediction of four transmembrane domains and seven short hydrophobicregions suggests that the PTRP protein is also likely to be a cellsurface protein.

Recent analysis of ˜4000 open reading frames from the genome sequence topredict their subcellular location showed that in contrast to B.subtilis, Mtb has 4-fold more proteins with extremely basic pIs (42). Incontrast, all 4 proteins identified in this study have acidic pIsranging between 4-5. Since the ERP and the MtrA are known to beexpressed during intracellular residence (3, 44), these observationsraise the possibility that the PTRP and the PE-PGRS protein identifiedin this study may also be expressed (or upregulated) under similarconditions. This hypothesis is further strengthened by the observationthat pre-TB sera had antibodies to both the proteins. Since the pre-TBsera were obtained from the patients 6 months prior to clinicalmanifestation of TB, and since none of these patients had cavitarylesions even at the time of clinical confirmation of TB, the bacteriareplication would be intracellular during the pre-TB stage in thesepatients.

It is also interesting that 3 of the 4 antigens identified in this studyare repetitive proteins. Proteins with tandem repetitive motifs arefound in several eukaryotic (1, 20, 23, 27, 35) and prokaryoticorganisms (13, 16, 17). In fact, a vast majority of gram-positive cellwall associated proteins have tandem repeats of amino acid sequences,which are associated with binding domains for host cell ligands. In manyinstances, the ability to alter the numbers of the repetitive domainscontributes to antigenic variation and to adapting to environmentalchanges (22). Many of the repetitive proteins are anchored on thecell-wall by the C terminal region containing the LPXTGX motif, butothers that may be anchored by charge and/or hydrophobic interactionshave been reported (15). The C-terminal portion of another member of thePE-PGRS family (Rv1759c) of Mtb has recently been shown to bindfibronectin (14), and an M. leprae 21 kD surface protein with 11 repeatsof XKKX motif at the C-terminal has been shown to bind the laminin-2 ofperipheral nerves, thus facilitating the entry of the bacilli intoSchwann cells. (38). In addition, the heparin binding hemagglutinin(HBHA) of Mtb that has been shown to be an adhesin which binds toepithelial cells via the Pro/lys repeats in the C-terminal region (31,33). The PTRP (Rv 0538) is structurally similar to these proteins inhaving the repetitive regions clustered in the C-terminal region,suggesting that it may have a similar fUnction.

The PE-PGRS (Rv3367) protein belongs to the PE family of proteins whichis one of the two large, clustered multigene families of glycine-richacidic proteins discovered when the genome sequence of Mtb wasdetermined (9). Some information is now available regarding expression,subcellular location and function of the PE_PGRS family proteins (14,34). Thus, the fibronectin-binding PE-PGRS protein encoded by Rv 1759c(described above) has been reported to be absent from antigenpreparations made from bacteria grown in bacteriological media (14),although the presence of antibodies in patient sera confirm its in vivoexpression. Also, PE-PGRS proteins of M. marinum, homologous to MtbPE-PGRS proteins (Rv3812 and Rv1651c) have been shown to be induced incultured macrophages as well as in frog granulomas (34). Although, noprotein band of the molecular weight corresponding to the PE-PGRS(Rv3367) protein (49 kDa) was observed in the LFCFP and SDS-CW (FIG.13), whether this protein is really not expressed during in vitrogrowth, or is expressed very poorly, or is destroyed during thepreparation of the LFCFP and the SDS-CWP remains to be determined.

The presence of antibodies in sera from TB patients to all the fourproteins identified, and their absence in the sera from PPD positivehealthy individuals shows that these proteins are expressed by the invivo Mtb only during active infection in humans. The mtrA promoter hasearlier been shown to be upregulated/activated upon entry and incubationof Mtb in macrophages (44) and the presence of anti MtrA antibodies inpre-TB and non-cavitary TB sera suggests that it is expressed in vivoduring intracellular bacterial replication. The β-gal fusion proteins ofPE_PGRS and PTRP were also well recognized by the pre-TB sera. We haveearlier shown that an 88 kDa culture filtrate protein is recognized byantibodies in the pre-TB sera of about 75% of the HIV-infected TBpatients (24). Thus, along with the 88 kDa protein, these 3 proteins maybe useful for developing surrogate markers for identifying HIV and Mtbco-infected individuals who are at a high risk of reactivating latentTB. Such markers have the potential to make significant contribution totuberculosis control in countries with high incidence of co-infection.

Earlier studies have shown that antibodies to the ERP homologs arepresent in M. bovis infected cattle, and in leprosy patients (5). Ourresults show that cavitary TB patients have antibodies to β-gal fusionprotein of the ERP, but the sera from non-cavitary TB patients and thepre-TB sera did not show reactivity even when individual patients weretested (data not shown). It is possible that in the human tissueenvironment, this protein is not well-expressed, and therefore isimmunogenic only when the bacterial load is high.

In summary, we have identified 4 antigenic proteins of Mtb that areimmunodominant during the early phase of an active Mtb infection. Allthe antigens appear to be surface proteins, and their involvement inbacillary adhesion and/or invasion is currently under investigation.Three of the 4 antigens are potential candidates for devisingimmunodiagnostic tests for identification of individuals with active,sub-clinical TB. Since many antigens of Mtb, including those that haveprovided some degree of protection in animal models, have been reportedto elicit both cellular and humoral immune responses (2, 12, 19, 43),and since these antigens are expressed in rabbits at the time whencellular immune responses that restrict bacterial growth of the inhaledbacteria are elicited, they are also being studied for their inclusionas components of a subunit vaccine for TB.

References Cited in Examples VI-XIII

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11 Dannenberg, A. M., Jr. 1991. Delayed-type hypersensitivity and cellmediated immunity in the pathogenesis of immunity. Immunol. Today.12:228-233.

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13 Drmsi, S., P. Dehoux, and P. Cossart. 1993. Common features ofGram-positive proteins involved in cell recognition. Mol. Microbiol.9:1119-1122.

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15 Fischetti, V. A. 2000. Surface Proteins on Gram-Positive Bacteria, p.11-24. In A. S. f. Microbiology (ed.), Gram-Positive Pathogens,Washinton, D.C.

16 Fischetti, V. A., M. Jarymowycz, K. Jones, and J. R. scott. 1986.Streptococcal M protein size mutants occur at high frequency within asingle strain. J. Exp. Med. 164:971-980.

17 Gaillard, J. L., P. Berche, C. Frehel, E. Goulin, and P. Cossart.1991. Entry of L. monocytogenes into cells is mediated by internalin, arepeat protein reminiscent of surface antigens from gram-positive cocci.Cell. 65:1127-1141.

18 Garbe, T. R., N. S. Hibler, and V. Deretic. 1999. Response toreactive nitrogen intermediates in Mycobacterium tuberculosis: inductionof the 16 kilodalton alpha-crystallin homolog by exposure to nitricoxide donors. Infect. Immun. 67:460-465.

19 Horwitz, M. A., B. W. E. Lee, B. J. Dillon, and G. Harth. 1995.Protective immunity against tuberculosis induced by vaccination withmajor extracellular proteins of Mycobacterium tuberculosis. Proc. Natl.Acad. Sci. USA. 92:1530-1534.

20 Ibanez, C. F., J. L. Affranchino, R. A. Macina, M. B. Reyes, S.Leguizamon, M. E. Camargo, L. Aslund, U. Pettersson, and A. C. C.Frasch. 1988. Multiple Trypanosoma cruzi antigens containing tandemlyrepeated amino acid sequence motifs. Mol. Bio. Parasitol. 30:27-34.

21 Isberg, R. R., and G. Tran Van Nhieu. 1994. Two mammalian cellinternalization strategies used by pathogenic bacteria. Annu. Rev.Genet. 27:395-422.

22 Kehoe, M. A. 1984. Cell-Wall-associated proteins in Gram-positvebacteria. In J.-M. Ghuysen and R. Hakenbeck (ed.), Bacterial Cell Wall,vol. Chapter 11. Elsevier Science B.V.

23 Kemp, D. J., R. L. Coppel, and R. F. Anders. 1987. Repetitiveproteins and genes of malaria. Ann. Rev. Microbiol. 41:181-208.

24 Laal, S., K. M. Samanich, M. G. Sonnenberg, J. T. Belisle, J.O'Leary, M. S. Simberkoff, and S. Zolla-Pazner. 1997. Surrogate markerof preclinical tuberculosis in human immunodeficiency virus infection:antibodies to an 88 kDa secreted antigen of Mycobacterium tuberculosis.J. Infect. Dis. 176:133-143.

25 Laal, S., K. M. Samanich, M. G. Sonnenberg, S. Zolla-Pazner, J. M.Phadtare, and J. T. Belisle. 1996. Human humoral responses to antigensof Mycobacterium tuberculosis: immunodominance of high molecular weightantigens. Clin. Diag. Lab. Imunnol. 4:49-56.

26 Lee, B. Y., and M. A. Horwitz. 1995. Identification of macrophage andstress-induced proteins of Mycobacterium tuberculosis. J. Clin. Invest.96:245-249.

27 Longacre, S., U. Hibner, A. Raibaud, H. Eisen, T. Baltz, C. Giroud,and D. Baltz. 1983. DNA rearangements and antigenic variation inTrypanosoma equiperdum: multiple expression-linked sites in independentisolates of Trypanosomes expressing the same antigen. Mol. Cell. Biol.3:399-409.

28 Lurie, M. Chapter VIII/ Host-Parasite Relations in Natively Resistantand Susceptible Rabbits on Quantitive Inhalation of Human and BovineTubercle Bacilli, and Nature of Genetic Resistance to Tuberculosis., p.192-222, Resistance to Tuberculosis; Experimental Studies in native andAcquired Defensive Mechanisms. Harvard University Press, Cambridge,Mass.

29 Lurie, M. B., and A. M. Dannenberg Jr. 1965. Macrophage finction inInfectious Disease with Inbred Rabbits. Bacterial Reviews. 29:466-475.

30 Marques, M. A. M., S. Chitale, P. J. Brennan, and M. C. V. Pessolani.1998. Mapping and identification of the Major Cell Wall-associatedcomponents of Mycobacterium laprae. Infect. and Immunity. 66:2625-2631.

31 Menozzi, F. D., R. Bischoff, E. Fort, M. J. Brennan, and C. Locht.1998. Molecular characterization of the mycobacterial heparin-bindinghemagglutinin, a mycobacterial adhesin. Proc. Natl. Acad. Sci. USA.95:12625-12630.

32 Patti, J. M., B. L. Allen, M. J. McGavin, and M. Hook. 1994.MSCRAMN-Mediated Adherence of Microorganisms to Host Tissues. Annu. Rev.Microbiol. 48:585-617.

33 Pethe, K., M. Aumercier, E. Fort, C. Gatot, C. Locht, and F. D.Menozzi. 2000. Characterization of the Heparin-binding site of theMycobacterial Heparin-binding Hemagglutinin Adhesin. The Journal ofBiological Chemistry. 275:14273-14273.

34 Ramakrishnan, L., N. A. Federspiel, and S. Falkow. 2000.Granuloma-Specific Expression of Mycobacterium Virulence proteins fromthe glycine-rich PE-PGRS family. Science. 288:1436-1439.

35 Richardson, J. P., R. P. Beecroft, D. L. Tolson, M. K. Liu, and T. W.Pearson. 1988. Procyclin: an unusual immunodominant glycoprotein surfaceantigen from the procyclic stage of African trypanosomes. Mol. Biochem.Parasitol. 31:203-216.

36 Samanich, K. M., J. T. Belisle, M. G. Sonnenberg, M. A. Keen, S.Zolla-Pazner, and S. Laal. 1998. Delineation of human antibody responsesto culture filtrate antigens of Mycobacterium tuberculosis. J. Infect.Dis. 178:1534-1538.

37 Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

38 Shimoji, Y., V. Ng, K. Matsumura, V. A. Fischetti, and A. Rambukkana.1999. A 21-kDa surface protein of Mycobacterium leprae binds peripheralnerve laminin-2 and mediates Schwann cell invasion. Proc. Natl. Acad.Sci. 96:9857-9862.

39 Smith, I., J. Dubnau, R. Manganelli, G. M. Rodriguez, B. Gold, S.Walters, J. Chan, and W. Rom. 1999. Identification and Characterizationof Potential Virulence genes of Mycobacterium tuberculosis, p. 108-112.US-Japan Cooperative Medical Science Program-Thirty-FourthTuberculosis-Leprosy Research Conference, San Francisco-Calif.

40 Smith, I., O. Duserget, M. Rodriquez, J. Timm, M. Gomez, J. Dubnau,B. Gold, and R. manganelli. 1998. Extra and intracellular expression ofMycobacterium tuberculosis genes. Tubercle and Lung Disease. 79:91-97.

41 Sonnenberg, M. G., and J. T. Belisle. 1997. Definition ofMycobacterium tuberculosis culture filtrate proteins by two-dimensionalpolyacrylamide gel electrophoresis, N-terminal amino acid sequencing andelectrospray mass spectrometry. Infect. Immun. 65:4515-4524.

42 Tekaia, F., S. V. Gordon, T. Garnier, R. Brosch, B. G. Barrell, andS. T. Cole. 1999. Analysis of the proteome of Mycobacterium tuberculosisin silico. Tubercle and Lung Disease. 79:329-342.

43 van Vooren, J. P., A. Drowart, M. de Cock, A. van Onckelen, D. H. M.H, J. C. Yernault, C. Valcke, and K. Huygen. 1991. Humoral immuneresponse of tuberculous patients against the three components of theMycobacterium bovis BCG 85 complex separated by isoelectric focusing. J.Clin. Microbiol. 29:2348-2350.

44 Via, L., R. C. M. Mudd, S. Dhandayuthapani, R. Ulmer, and V. Deretic.1996. Elements of signal transduction in mycobacterium tuberculosis: invitro phosphorylation and in vitro expression of the response regulatorMtrA. J. Bacteriology. 178:3314-21.

45 Wong, D. K., B.-Y. Lee, M. A. Horwitz, and B. W. Gibson. 1999.Identification of fur, aconitase, and other proteins expressed byMycobacteriurn tuberculosis under conditions of low and highconcentrations of iron by combined two-dimensional gel electrophoresisand mass spectrometry. Infect. Immun. 67:327-336.

46 Young, D. B., and K. Duncan. 1995. Prospects for new interventions inthe treatment and prevention of mycobacterial diease. Annu. Rev.Microbiol. 49:641-673.

47 Young, R. A., B. R. Bloom, C. M. Grosskinsky, J. Ivannyi, D. Thomas,and R. W. Davis. 1985. Dissection of Mycobacterium tuberculosis antigensusing recombinant DNA. Proc. Natl. Acad. Sci. USA. 82:2583-2587.

EXAMPLE XIV Definition of M. tuberculosis Culture Filtrate Proteins by2-Dimensional Polyacrylamide Gel Electrophoresis Mapping, N-terminalAmino Acid Sequencing and Electrospray Mass Spectrometry

This Example that describes various individual culture filtrate proteinsof Mtb is taken from U.S. Pat. No. 6,245,331 (12 Jun. 2001) which, asindicated, is incorporated by reference in its entirety. (See Example Vtherein)

The combination of 2-D PAGE, western blot analysis, N-terminal aminoacid sequencing and liquid chromatography-mass spectrometry-massspectrometry (LC-MS-MS) was used to develop a detailed map of culturefiltrate proteins and to obtained partial amino acid sequences for fivepreviously undefined, relatively abundant proteins within this fractionwhich are found to be useful as early antigens for serodiagnosis of TB.

These proteins were shown to be early antigens of TB recognized bycirculating antibodies in TB patients early in the disease process.

SDS-PAGE and 2-D PAGE of Culture Filtrate Proteins

SDS-PAGE was performed under reducing conditions by the method ofLaemmli with gels (7.5×10 cm×0.75 mm) containing a 6% stack over a 15%resolving gel. Each gel was run at 10 mA for 15 min followed by 15 mAfor 1.5 h.

2-D PAGE separation of proteins was achieved by the method of O'Farrellwith minor modifications. Specifically, 70 μg of CFP was dried andsuspended in 30 μl of isoelectric focusing (IEF) sample buffer [9 Murea, 2% Nonidet P-40, 5% βmercaptoethanol, and 5% ampholytes pH 3-10(Pharmalytes; Pharmacia Biotech, Piscataway, N.J.)], and incubated for 3h at 20° C. An aliquot of 25 μg of protein was applied to a 6%polyacrylamide IEF tube gel (1.5 mm by 6.5 cm) containing 5% PharmalytespH 3-10 and 4-6.5 in a ratio of 1:4. The proteins were focused for 3 hat 1 kV using 10 mM H₃PO₄ and 20 mM NaOH as the catholyte and anolyte,respectively. The tube gels were subsequently imbibed in sample transferbuffer for 30 min and placed on a preparative SDS-polyacrylamide gel(7.5×10 cm×1.5 mm) containing a 6% stack over a 15% resolving gel.Electrophoresis in the second dimension was carried out at 20 mA per gelfor 0.3 h followed by 30 mA per gel for 1.8 h. Proteins were visualizedby staining with silver nitrate.

Silver stained 2-D PAGE gels were imaged using a cooled CCD digitizingcamera and analyzed with MicroScan 1000 2-D Gel Analysis Software forWindows 3.x (Technology Resources, Inc., Nashville, Tenn.). Protein peaklocalization and analysis was conducted with the spot filter on, aminimum allowable peak height of 1.0, and minimum allowable peak area of2.0.

Proteins, subjected to 2-D or SDS-PAGE, were transferred tonitrocellulose membranes (Schleicher and Schuell, Keene, N.H.) whichwere blocked with 0.1% bovine serum albumin in 0.05 M Tris-HCl, pH 7.5,0.15 M NaCl, and 0.05% Tween 80 (TBST). These membranes were incubatedfor 2 h with specific antibodies diluted with TBST to the proper workingconcentrations. After washing, the membranes were incubated for 1 h withgoat anti-mouse or -rabbit alkaline phosphatase-conjugated antibody(Sigma) diluted in TBST. The substrates nitro-blue-tetrazolium and5-bromo-4-chloro-3-indoyl phosphate (BCIP) were used for colordevelopment.

Mapping of proteins reactive to specific antibodies within the 2-D PAGEgel was accomplished using 0.1% India ink as a secondary stain for thetotal protein population after detection by immunoblotting.Alternatively, the Digoxigenin (DIG) Total Protein/Antigen DoubleStaining Kit (Boehringer Mannheim, Indianapolis, Ind.) was employed forthose antibody-reactive proteins that could not be mapped using Indiaink as the secondary stain. Briefly, after electroblotting, themembranes were washed three times in 0.05 M K₂HPO₄, pH 8.5. The totalprotein population was conjugated to digoxigenin by incubating themembrane for one hour at room temperature in a solution of 0.05 MK₂HPO₄, pH 8.5 containing 0.3 ng/mldigoxigenin-3-0-methylcarbonyl-ε-amino-caproic acid N-hydroxysuccinimideester and 0.01% Nonidet-P40. The membranes were subsequently blockedwith a solution of 3% bovine serum albumin in 0.05 M Tris-HCl, pH 7.5,0.15 M NaCl (TBS) for 1 h followed by washing with TBS. Incubation withspecific antibodies was performed as described, followed by incubationof the membranes with mouse anti-DIG-Fab fragments conjugated toalkaline phosphatase diluted 1:2000 in TBS, for 1 h. The membranes werewashed three times with TBS and probed with goat anti-mouse or -rabbithorse radish peroxidase-conjugated antibody. Color development for theproteins reacting to the specific anti-Mtb protein antibodies wasobtained with the substrates 4-(1,4,7,10-tetraoxadecyl)-1-naphthol and1.8% H₂O₂. Secondary color development of the total protein populationlabeled with digoxigenin utilized BCIP and[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride] asthe substrates.

To obtain N-terminal amino acid sequence for selected proteins, CFPs(200 μg) were resolved by 2-D PAGE and transferred to polyvinylidenedifluoride membrane (Millipore, Milford, Mass.) by electroblotting at 50V for 1 h, using CAPS buffer with 10% methanol. The membrane was stainedwith 0.1% Coomassie brilliant blue in 10% acetic acid and destained witha solution of 50% methanol and 10% acetic acid. Immobilized proteinswere subjected to automated Edman degradation on a gas phase sequencerequipped with a continuous-flow reactor. The phenylthiohydantoin aminoacid derivatives were identified by on-line reversed-phasechromatography as described previously.

Selected CFP were subjected to LC-MS-MS to determine the sequence ofinternal peptide fragments. CFPs (200 mg) were resolved by 2-D PAGE andthe gel stained with 0.1% Coomassie brilliant blue and destained asdescribed for proteins immobilized to PVDF membranes. The protein ofinterest was excised from the gel, washed several times with distilledwater to remove residual acetic acid and subjected to in-gel proteolyticdigestion with trypsin. Peptides were eluted from the acrylamide andseparated by C18 capillary RP-HPLC. The microcapillary RP-HPLC effluentwas introduced directly into a Finnigan-MAT (San Jose, Calif.) TSQ-700triple sector quadrupole mass spectrometer. Mass spectrometry andanalysis of the data was performed as described by Blyn et al..

C. Results

1. Definition of Proteins Present in the Culture Filtrate of Mtb H37Rv.

Through the efforts of the World Health Organization (WHO) ScientificWorking Groups (SWGs) on the Immunology of Leprosy (IMMLEP) andImmunology of Tuberculosis (IMMTUB) an extensive collection of mAbsagainst mycobacterial proteins has been established. This library aswell as mAbs and polyclonal sera not included in these collectionsallowed for the identification of known mycobacterial proteins in theculture filtrate of Mt. A detailed search of the literature identifiedmAbs and/or polyclonal sera reactive against 35 individual Mtb CFP(Table 1). Initially, the presence or absence of these proteins in theculture filtrate of Mtb H37Rv, prepared for these studies, wasdetermined by Western blot analyses. Of the antibodies and sera tested,all but one (IT-56) demonstrated reactivity to specific proteins of thispreparation (Table 1). The mAb IT-56 is specific for the 65 kDa MtbGroEL homologue; a protein primarily associated with the cytosol.Additionally the mAb IT-7 reacted with a 14 kDa and not a 40 kDa CFP.

2. 2-D PAGE Mapping of Known CFP of Mtb H37Rv

Using 2-D western blot analysis coupled with secondary staining (eitherIndia ink or Dig total protein/antigen double staining) the proteinsreactive to specific mAbs or polyclonal sera were mapped within the 2-DPAGE profile of CFP of Mtb H37Rv. In all, 32 of the reactive antibodiesdetected specific proteins resolved by 2-D PAGE (Table 1). However, twoantibodies (IT-1 and IT-46), that were reactive by conventional westernblot analysis, failed to detect any protein within the 2-D profile (notshown; summarized in Tables). This lack of reactivity by 2-D westernanalysis, presumably, was due to the absence of linear epitopes exposedby the denaturing conditions used to resolve molecules for conventionalWestern blot analyses.

The majority of the antibodies recognized a single protein spot.However, several (IT-3, IT-4, IT-7, IT-20, IT-23, IT-41, IT-42, IT-44,IT-49, IT-57, IT-58, IT-61 and MPT 32) reacted with multiple proteins.Five of these, IT-23, IT-42, IT-44, IT-57 and IT-58 reacted with proteinclusters centered at 36 kDa, 85 kDa, 31 kDa, 85 kDa and 50 kDa,respectively. Additionally the proteins in each of these clustersmigrated within a narrow pI range; suggesting that the antibodies werereacting with multiple isoforms of their respective proteins. In thecase of the protein cluster at 85 kDa (which is the “88 kDa” identifiedas malate synthase) detected by IT-57, the most dominant component ofthis cluster was also recognized by IT-42.

Polyclonal sera against MPT 32 recognized a 45 and 42 kDa protein ofrelatively similar pI. While defining sites of glycosylation on MPT 32(see above) we observed that this protein was prone to autoproteolysisand formed a 42 kDa product. Thus, the 42 kDa protein detected with theanti-MPT 32 sera was a breakdown product of the 45 kDa MPT 32glycoprotein. The mAb (T-49 specific for the Antigen 85 (Ag85) complexclearly identified the three gene products (Ag85A, B and C) of thiscomplex. The greatest region of antibody cross-reactivity was atmolecular masses below 16 kDa. The most prominent protein in this regionreacted with niAb IT-3 specific for the 14 kDa GroES homolog. This mAbalso recognized several adjacent proteins at approximately 14 kDa.Interestingly, various members of this same protein cluster reacted withanti-MPT 57 and anti-MPT 46 polyclonal sera, and the mAbs IT-4, IT-7,and IT-20.

3. N-terminal Amino Acid Sequencing of Selected CFPs

The N-terminal amino acid sequences or complete gene sequences andfunctions of several of the CFPs of Mt, mapped with the availableantibodies, are known. However, such information is lacking for theproteins that reacted with IT-42 IT-43, IT-44, IT-45, IT-51, IT-52,IT-53, IT-57, IT-59 and IT-69, as well as several dominant proteins notidentified by these means. Of these, the most abundant proteins (IT-52,IT-57, IT 42, IT-58 and proteins labeled A-K) were selected andsubjected to N-terminal amino acid sequencing. TABLE 1 Reactivity ofCFPs of M. tuberculosis H₃₇Rv to reported specific mAbs and polyclonalantisera Dilution REACTIVITY Antibody¹ MW (kDa) Used 1-D 2-D IT-1(F23-49-7) 16 kDa  1:2000 + − IT-3 (SA-12) 12 kDa  1:8000 + + IT-4(F24-2-3) 16 kDa  1:2000 + + IT-7 (F29-29-7) 40 kDa  1:1000 + + IT-10(F29-47-3) 21 kDa  1:1000 + + IT-12 (HYT6) 17-19 kDa 1:50 + + IT-17(D2D) 23 kDa  1:8000 + + IT-20 (WTB68-A1) 14 kDa  1:250 + + IT-23(WTB71-H3) 38 kDa  1:250 + + IT-40 (HAT1) 71 kDa 1:50 + + IT-41 (HAT3)71 kDa 1:50 + + IT-42 (HBT1) 82 kDa 1:50 + + IT-43 (HBT3) 56 kDa1:50 + + IT-44 (HBT7) 32 kDa 1:50 + + IT-45 (HBT8) 96 kDa 1:50 + + IT-46(HBT10) 40 kDa 1:50 + − IT-49 (HYT27) 32-33 kDa 1:50 + + IT-51 (HBT2) 17kDa 1:50 + + IT-52 (HBT4) 25 kDa 1:50 + + IT-53 (HBT5) 96 kDa 1:50 + +IT-56 (CBA1) 65 kDa 1:50 − ND* IT-57 (CBA4) 82 kDa 1:50 + + IT-58 (CBA5)47 kDa 1:50 + + IT-59 (F67-1) 33 kDa  1:100 + + IT-61 (F116-5) 30 (24)kDa  1:100 + + IT-67 (L24.b4) 24 kDa 1:50 + + IT-69 (HBT 11) 20 kDa1:6  + + F126-2 30 kDa  1:100 + + A3h4 27 kDa 1:50 + + HYB 76-8 6 kDa 1:100 + + anti-MPT 32 50 kDa  1:100 + + anti-MPT 46 10 kDa  1:100 + +anti-MPT 53 15 kDa  1:100 + + anti-MPT 57 12 kDa  1:100 + + anti-MPT63 - K64 18 kDa  1:200 + +*ND: Not done¹Original designations for the World Health Organization cataloged Mabare given in parentheses.

Three of these proteins were found to correspond to previously definedproducts. The N-terminal amino acid sequence of the protein labeled Dwas identical to that of Ag85 B and C. This result was unexpected giventhat the IT-49 mAb failed to detect this protein and N-terminal aminoacid analysis confirmed that those proteins reacting with IT-49 weremembers of the Ag85 complex. Second, the protein labeled E had anN-terninal sequence identical to that of glutamine synthetase. A thirdprotein which reacted with IT-52 was found to be identical to MPT 51.

However, five of the proteins analyzed appeared to be novel. Three ofthese, those labeled B, C and IT-58 did not demonstrate significanthomology to any known mycobacterial or prokaryotic sequences. Theprotein labeled I possessed an N-terminal sequence with 72% identity tothe amino terminus of an α-hydroxysteroid dehydrogenase from aEubacterium species, and the protein labeled F was homologous to adeduced amino acid sequence for an open reading frame identified in theMtb cosmid MTCY1A11. Repeated attempts to sequence those proteinslabeled as A, G, H, J, K, IT-43, IT-44, IT-49 and IT-57 wereunsuccessful.

Reactivity of Tuberculosis Sera with the M. tuberculosis 88 kDa Antigen

A high molecular weight fraction of CFP of Mtb reacted with apreponderance of sera from TB patients and that this fraction wasdistinguished from other native fractions in that it possessed theproduct initially thought to be reactive to mAb IT-57. In view of this,the protein cluster (the 88 kDa protein) initially thought to be definedby IT-42 and IT-57 was excised from a 2-D polyacrylamide gel, digestedwith trypsin and the resulting peptides analyzed by LC-MS-MS. In orderto confirm that M. tuberculosis also contains a seroreactive 88 kDaantigen which is not the catalase/peroxidase, a katG-negative strain ofM. tuberculosis (ATCC 35822) was tested. Lysates from this strain failedto react with any of the anti-catalase/peroxidase antibodies However,when individual sera from healthy controls and TB patients of all threegroups were tested with the same lysates, all the group III and group IVsera reacted with the 88 kDa protein

Identification of the Amino Acid Sequence of the Sero-Reactive 88 kDaProtein

The culture filtrate protein from a katG-negative strain of Mtuberculosis (ATCC 35822) was resolved as above by 2-D PAGE. The proteinspot corresponding to the sero-reactive 88 kDa protein was cut out ofthe gel and subject to an in-gel digestion with trypsin. The resultingtryptic peptides were exteracted, applied to a C₁₈ RP-HPLC column, andeluted with an increasing concentration of acetonitrile. The peptideseluted in this manner were introduced directly into a Finnigan LCQElectrospray mass spectrometer. The molecular mass of each peptide wasdetermined, as was the charge state, with a zoom-scan program.Identification of the 88 kDa protein was achieved by entering the massspectroscopy date obtained above into the MS-Fit computer program andsearching it against the M. tuberculosis database.

The protein was identified as GlcB (Z78020) of M. tuberculosis, which isbelieved to be the enzyme malate synthase based on sequence homology toknown proteins of other bacteria This protein has the Accession numberCAB01465 on the NCBI Genbank database (based on Cole, S. T. et al.,Nature 393:537-544 (1998), which describes the complete genome sequenceof M. tuberculosis). The sequence of this protein, SEQ ID NO: 13 ispresented below.

C. Discussion

In contrast to Mtb cell wall, cell membrane and cytoplasmic proteins,the CFPs are well defined in terms of function, immunogenicity andcomposition. However, a detailed analysis of the total proteins, and themolecular definition and 2-D PAGE mapping of the majority of these CFPshas not been performed. Nagai and colleagues identified and mapped by2-D PAGE the most abundant proteins filtrate harvested after five weeksof culture in Sauton medium. The present study used culture filtratesfrom mid- to late-logarithmic cultures of three Mtb type strains H37Ra,H37Rv, and Erdman to provide for the first time a detailed analysisunderstanding of this widely studied fraction.

Computer analysis of the 2-D gels of CFP resolved 205, 203 and 206individual protein spots from filtrates of strains H37Rv, H37Ra andErdman, respectively. Of the total spots, 37 were identified using acollection of mAb and polyclonal sera against CFPs. Several of theseantibodies recognized more than one spot; several are believed to reactwith multiple isoforms of the same protein or were previously shown torecognize more then a single gene product. In all, partial or completeamino acid sequences have been reported for 17 of the proteins mappedwith the available antibodies.

For greater molecular definition, a number of abundant products observedin the 2-D PAGE were subjected to N-terminal sequence analysis.

One such protein that migrated between Ag85B and Ag85C was found to have16 residues (FSRPGLPVEYLQVPSP, [SEQ ID NO:12]) identical to theN-terminus of mature Ag85A and Ag85B, and different from Ag85C by asingle residue (position 15). This protein spot was apparently merely ahomologue of Ag85A or B. However, its complete lack of reactivity withan Ag85-specific mAb (IT-49), its weight greater than that of Ag85B andits shift in pI in relation to Ag85A suggested that this product mayhave resulted from post translational modifications. Alternatively, thisprotein may be a yet unrecognized fourth member of the Ag85 complex.However, members of the Ag85 complex appear to lack post-translationalmodifications in some reports whereas others report several bandscorresponding to Ag85C after isoelectric focusing. However, no directevidence supports the existence of a fourth Ag85 product.

A second product sequenced was a 25 kDa protein with a pI of 5.34. ItsN-terminal sequence (XPVM/LVXPGXEXXQDN, [SEQ ID NO:15]) showed homologyto an internal fragment (DPVLVFPGMEIRQDN, [SEQ ID NO: 16]) correspondingto open reading frame 28c of the Mtb cosmid MTCY1A11. Analysis of thatdeduced sequence revealed a signal peptidase I consensus sequence(Ala-Xaa-Ala) and an apparent signal peptide preceding the N-terminus ofthe 25 kDa protein sequenced above

N-terminal sequencing of selected CFPs identified three novel products:

-   (I) protein with 72% identity to the N-terminus of a 42 kDa    α-hydroxysteroid dehydrogenase of Eubacterium sp. VPI 12708;-   (2) 27 kDa protein previously defined as MPT-51; and-   (3) 56 kDa protein previously identified as glutamine synthetase.

Three proteins showed no significant homology between their N-terminiand any known peptides. For these proteins and for others that wererefractory to N-group analysis, more advanced methods of proteinsequencing (e.g., LC-MS-MS) will permit acquisition of extended sequenceinformation.

This type of broad survey of virulent Mtb strains has led to, and willcontinue to allow, the identification of immunologically importantproteins and will lead to identification of novel virulence factorsleading to improved approaches to chemotherapy. Thus, not only does thepresent invention enhance the overall knowledge in the art of thephysiology of Mt, but it also provides immediate tools for earlyserodiagnosis. TABLE 2 Summary of certain protein spots detected bycomputer aided analysis of silver nitrate stained 2-D gels. AntibodyFunction/ N-terminal Ref #. H37Rv H37Ra Erdman MW(kDa) pI ReactivityDesignation Sequence SEQ ID NO 11 11 11 11  38.90 4.31 anti-MPT 32 MPT32 DPAPAPPVPT 9 14 14 14 14  42.17 4.51 anti-MPT 32 MPT 32 DPAPAPPVPT 924 24 24 24  48.70 4.79 59 59 59 59  29.68 5.08 66 66 66 66  35.69 5.09IT-23 PstS CGSKPPSPET 10 68 68 68 68  42.41 5.10 69 69 69 69  30.20 5.1077 77 77 77  28.18 5.10 80 80 80 80  42.17 5.10 C XXAVXVT 11 103 103 103103  31.08 5.12 D: Antigen 85 FSRPGLPVEYLQVPSP 12 Homolog? 111 111 111111 104.71 5.13 124 124 124 124  85.11 (88) 5.19 Malate synthase Seebelow for full 13 sequencel 170 170 170 170  26.92 5.91 IT-52 MPT 51 Seebelow for full 14 sequence

Amino Acid Sequence of 88 kDa Malate Synthase (SEQ ID NO: 13):MTDRVSVGNL RIARVLYDFV NNEALPGTDI DPDSFWAGVD KVVADLTPQN QALLNARDELQAQIDKWHRR RVIEPIDMDA YRQFLTEIGY LLPEPDDFTI TTSGVDAEIT TTAGPQLVVPVLNARFALNA ANARWGSLYD ALYGTDVIPE TDGAEKGPTY NKVRGDKVIA YARKFLDDSVPLSSGSFGDA TGFTVQDGQL VVALPDKSTG LANPGQFAGY TGAAESPTSV LLINHGLHIEILIDPESQVG TTDRAGVKDV ILESAITTIM DFEDSVAAVD AADKVLGYRN WLGLNKGDLAAAVDKDGTAF LRVLNRDRNY TAPGGGQFTL PGRSLMFVRN VGHLMTNDAI VDTDGSEVFEGIMDALFTGL IAIHGLKASD VNGPLINSRT GSIYIVKPKM HGPAEVAFTC ELFSRVEDVLGLPQNTMKIG IMDEERRTTV NLKACIKAAA DRVVFINTGF LDRTGDEIHT SMEAGPMVRKGTMKSQPWIL AYEDHNVDAG LAAGFSGRAQ VGKGMWTMTE LMADMVETKI AQPRAGASTAWVPSPTAATL HALHYHQVDV AAVQQGLAGK RRATIEQLLT IPLAKELAWA PDEIREEVDNNCQSILGYVV RWVDQGVGCS KVPDIHDVAL MEDRATLRIS SQLLANWLRH GVITSADVRASLERMAPLVD RQNAGDVAYR PMAPNFDDSI AFLAAQELIL SGAQQPNGYT EPILHRRRREFKARAAEKPA PSDRAGDDAA R

Amino Acid Sequence of Secreted Form of MPT 51 (SEQ ID NO:14):APYENLMVPS PSMGRDIPVA FLAGGPHAVY LLDAFNAGPD VSNWVTAGNA NTLAGKGISVVAPAGGAYS MYTNWEQDGS KQWDTFLSAE LPDWLAANRG AAQGGYGAMA AAFHPDRFGFAGSMSGFLY PSNTTTNGAI AAGMQQFGGV DTNGMWGAPQ LGRWKWHDPW HASLLAQNNTRVWVWSPTN PGASDPAAMI GQTAEAMGNS RMFYNQYRSV GGHNGHFDFP SGDNGWGSWAPQLGAMSGD IVGAIR.

The references cited above are all incorporated by reference herein,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

1. A method for the early detection of mycobacterial disease orinfection in a subject, comprising assaying a biological fluid samplefrom a subject having symptoms of active tuberculosis, but before theonset of symptoms identifiable as advanced tuberculosis for the presenceof early antibodies specific for one or more early Mtb antigens whichantigens are characterized as being surface or secreted proteins thatare (i) reactive with antibodies found in tuberculosis patients who arein a stage of disease prior to the onset of (a) smear-positivity ofsputum or other pulmonary associated fluid for acid-fast bacilli and (b)cavitary pulmonary lesions, and (ii) non-reactive with sera from healthycontrol subjects or healthy subjects with latent inactive tuberculosiswherein the presence of said early antibodies specific for said earlyantigens is indicative of the presence of said disease or infection. 2.A method for the early detection of mycobacterial disease or infectionin a subject, comprising assaying a biological fluid sample from asubject having symptoms of active tuberculosis, but before the onset ofsymptoms identifiable as advanced tuberculosis for the presence ofantibodies or T lymphocytes specific for or reactive with an early Mtbantigen selected from the group consisting of (a) PirG protein encodedby the Mtb gene Rv3810; (b) PE-PGRS protein encoded by the Mtb geneRv3367; (c) PTRP protein encoded by the Mtb gene Rv0538); (d) MtrAprotein encoded by the Mtb gene Rv3246c; and (e) an epitope of any of(a)-(d).
 3. A method for the early detection of mycobacterial disease orinfection in a subject, comprising assaying a biological fluid or cellor tissue sample from a subject having symptoms of active tuberculosis,but before the onset of symptoms identifiable as advanced tuberculosisfor the presence of one or more early M. tuberculosis early antigensselected from the group consisting of (a) PirG protein encoded by theMtb gene Rv3810;, (b) PE-PGRS protein encoded by the Mtb gene Rv3367;(c) PTRP protein encoded by the Mtb gene Rv0538); (d) MtrA proteinencoded by the Mtb gene Rv3246c; and (e) an epitope of any of (a)-(d),using an antiserum or a monoclonal antibody specific for an epitope ofsaid an early antigen, wherein the presence of said one or more earlyantigens is indicative of the presence of said disease or infection. 4.A method for the early detection of mycobacterial disease or infectionin a subject, comprising assaying a biological fluid sample from asubject having symptoms of active tuberculosis, but before the onset ofsymptoms identifiable as advanced tuberculosis for the presence ofimmune complexes consisting of one or more early M. tuberculosisantigens complexed with an antibody specific for said antigen selectedfrom the group consisting of (a) PirG protein encoded by the Mtb geneRv3810; (b) PE-PGRS protein encoded by the Mtb gene Rv3367; (c) PTRPprotein encoded by the Mtb gene Rv0538); and (d) MtrA protein encoded bythe Mtb gene Rv3246c, (e) an epitope of any of (a)-(d), wherein thepresence of said immune complexes is indicative of the presence of saiddisease or infection.
 5. The method of any one of claims 1-4 thatfurther includes performance of a test that detects mycobacterialbacilli in a sample of sputum or other body fluid of said subject. 6.The method of any of claims 1-5 wherein said biological fluid sample isserum, urine or saliva.
 7. The method of any of claims 1-6 comprising,prior to said assaying step, the step of removing from said sampleantibodies specific for cross-reactive epitopes or antigens of proteinspresent in M. tuberculosis and in other bacterial genera.
 8. The methodof any of claims 1-7 wherein said removing is performed byimmunoadsorption of said sample with E. coli antigens.
 9. The method ofany of claims 1-8, wherein said subject is a human.
 10. The method ofclaim 9 wherein said subject is infected with HIV-1 or is at high riskfor tuberculosis.
 11. The method of any of claims 1-10 which includesassaying said sample for antibodies specific for one or more additionalearly antigens of M. tuberculosis selected from the group consisting of:(a) an 88 kDa M. tuberculosis protein having the an amino acid sequenceSEQ ID NO:13: MTDRVSVGNL RIARVLYDFV NNEALPGTDI DPDSFWAGVD KVVADLTPQNQALLNARDEL QAQIDKWHRR RVIEPIDMDA YRQFLTEIGY LLPEPDDFTI TTSGVDAEITTTAGPQLVVP VLNARFALNA ANARWGSLYD ALYGTDVIPE TDGAEKGPTV NKVRGDKVIAYARKFLDDSV PLSSGSFGDA TGFTVQDGQL VVALPDKSTG LANPGQFAGY TGAAESPTSVLLINHGLHIE ILIDPESQVG TTDRAGVKDV ILESAITTIM DFEDSVAAVD AADKVLGYRNWLGLNKGDLA AAVDKDGTAF LRVLNRDRNY TAPGGGQFTL PGRSLMFVRN VGHLMTNDAIVDTDGSEVFE GIMDALFTGL IAIHGLKASD VNGPLINSRT GSIYIVKPKM HGPAEVAFTCELFSRVEDVL GLPQNTMKIG IMDEERRTTV NLKACIKAAA DRVVFINTGF LDRTGDEIHTSMEAGPMVRK GTMKSQPWIL AYEDHNVDAG LAAGFSGRAQ VGKGMWTMTE LMADMVETKIAQPRAGASTA WVPSPTAATL HALHYHQVDV AAVQQGLAGK RRATIEQLLT IPLAKELAWAPDEIREEVDN NCQSILGYVV RWVDQGVGCS KVPDIHDVAL MEDRATLRIS SQLLANWLRHGVITSADVRA SLERMAPLVD RQNAGDVAYR PMAPNFDDSI AFLAAQELIL SGAQQPNGYTEPILHRRRRE FKARAAEKPA PSDRAGDDAA R

(b) a 27 kDa M. tuberculosis protein named MPT51 having the amino acidsequence SEQ ID NO: 14: APYENLMVPS PSMGRDIPVA FLAGGPHAVY LLDAFNAGPDVSNWVTAGNA NTLAGKGIS VVAPAGGAYS MYTNWEQDGS KQWDTFLSAE LPDWLAANRGAAQGGYGAMA AAFHPDRFG FAGSMSGFLY PSNTTTNGAI AAGMQQFGGV DTNGMWGAPQLGRWKWHDPW HASLLAQNN TRVWVWSPTN PGASDPAAMI GQTAEAMGNS RMFYNQYRSVGGHNGHFDFP SGDNGWGSW APQLGAMSGD IVGAIR;

(c) a protein characterized as M. tuberculosis antigen 85C; and (d) aglycoprotein characterized as M. tuberculosis antigen MPT32.
 12. A kituseful for early detection of M. tuberculosis disease comprising: (a) anantigenic composition comprising one or more proteins selected from thegroup consisting of (i) PirG protein encoded by the Mtb gene Rv3810;(ii) PE-PGRS protein encoded by the Mtb gene Rv3367; (iii) PTRP proteinencoded by the Mtb gene Rv0538); and (iv) MtrA protein encoded by theMtb gene Rv3246c, or an epitope of any of (i)-(iv), in combination with(b) reagents necessary for detection of antibodies which bind to said M.tuberculosis protein.
 13. The kit of claim 12 further supplemented withone or more additional early antigens of M. tuberculosis selected fromthe group consisting of: (A) an 88 kDa M. tuberculosis protein havingthe an amino acid sequence SEQ ID NO:13: MTDRVSVGNL RIARVLYDFVNNEALPGTDI DPDSFWAGVD KVVADLTPQN QALLNARDEL QAQIDKWHRR RVIEPIDMDAYRQFLTEIGY LLPEPDDFTI TTSGVDAEIT TTAGPQLVVP VLNARFALNA ANARWGSLYDALYGTDVIPE TDGAEKGPTY NKVRGDKVIA YARKFLDDSV PLSSGSFGDA TGFTVQDGQLVVALPDKSTG LANPGQFAGY TGAAESPTSV LLINHGLHIE ILIDPESQVG TTDRAGVKDVILESAITTIM DFEDSVAAVD AADKVLGYRN WLGLNKGDLA AAVDKDGTAF LRVLNRDRNYTAPGGGQFTL PGRSLMFVRN VGHLMTNDAI VDTDGSEVFE GIMDALFTGL IAIHGLKASDVNGPLINSRT GSIYIVKPKM HGPAEVAFTC ELFSRVEDVL GLPQNTMKIG IMDEERRTTVNLKACIKAAA DRVVFINTGF LDRTGDEIHT SMEAGPMVRK GTMKSQPWIL AYEDHNVDAGLAAGFSGRAQ VGKGMWTMTE LMADMVETKI AQPRAGASTA WVPSPTAATL HALHYHQVDVAAVQQGLAGK RRATIEQLLT IPLAKELAWA PDEIREEVDN NCQSILGYVV RWVDQGVGCSKVPDIHDVAL MEDRATLRIS SQLLANWLRH GVITSADVRA SLERMAPLVD RQNAGDVAYRPMAPNFDDSI AFLAAQELIL SGAQQPNGYT EPILHRRRRE FKARAAEKPA PSDRAGDDAA R

(B) a 27 kDa M. tuberculosis protein named MPT51 having the amino acidsequence SEQ ID NO:14: APYENLMVPS PSMGRDIPVA FLAGGPHAVY LLDAFNAGPDVSNWVTAGNA MNTLAGKGIS VVAPAGGAYS MYTNWEQDGS KQWDTFLSAE LPDWLAANRGAAQGGYGAMA LAAFHPDRFG FAGSMSGFLY PSNTTTNGAI AAGMQQFGGV DTNGMWGAPQLGRWKWHDPW VHASLLAQNN TRVWVWSPTN PGASDPAAMI GQTAEAMGNS RMFYNQYRSVGGHNGHFDFP ASGDNGWGSW APQLGAMSGD IVGAIR

(C) a protein characterized as M. tuberculosis antigen 85C; or (D) aglycoprotein characterized as M. tuberculosis antigen MPT32.
 14. The kitof claim 12 or claim 13 further supplemented with one or more of thefollowing M. tuberculosis antigenic proteins having an approximatemolecular weight as indicated: (i) a 28 kDa protein corresponding to thespot identified as Ref. No. 77 in Table
 2. (ii) a 29/30 kDa proteincorresponding to the spot identified as Ref. No. 69 or 59 in Table 2;(iii) a 31 kDa protein corresponding to the spot identified as Ref. No.103 in Table 2; (iv) a 35 kDa protein corresponding to the spotidentified as Ref. No. 66 in Table 2 and reacting with monoclonalantibody IT-23; (v) a 42 kDa protein corresponding to the spotidentified as Ref. No. 68 or 80 in Table 2; (vi) a 48 kDa proteincorresponding to the spot identified as Ref. No. 24 in Table 2; and(vii) a 104 kDa protein corresponding to the spot identified as Ref. No.111 in Table 2, which spots are obtained by 2-dimensionalelectrophoretic separation of M. tuberculosis lipoarabinomannan-freeculture filtrate proteins as follows: (A) incubating 3 hours at 20° C.in 9M urea, 2% Nonidet P-40, 5% β-mercaptoethanol, and 5% anpholytes atpH 3-10; (B) isoelectric focusing on 6% polyacrylamide isoelectricfocusing tube gel of 1.5 mm×6.5 cm, said gel containing 5% ampholytes ina 1:4 ratio of pH 3-10 ampholytes to pH 4-6.5 ampholytes for 3 hours at1 kV using 10 mM H₃PO₄ as catholyte and 20 mM NaOH as anolyte, to obtaina focused gel; (C) subjecting the focused gel to SDS PAGE in the seconddimension by placement on a preparative SDS-polyacrylamide gel of 7.5×10cm×1.5 mm containing a 6% stack over a 15% resolving gel andelectrophoresing at 20 mA per gel for 0.3 hours followed by 30 mA pergel for 1.8 hours.
 15. The kit of any of claims according of claim 12wherein at least one of said early M. tuberculosis antigens is arecombinant protein or glycoprotein.
 16. An antigenic composition usefulfor early detection of M. tuberculosis disease or infection comprising aonr or a mixture of two or more early M. tuberculosis antigens whichantigens are selected from the group consisting of (a) PirG proteinencoded by the Mtb gene Rv3810; (b) PE-PGRS protein encoded by the Mtbgene Rv3367; (c) PTRP protein encoded by the Mtb gene Rv0538); (d) MtrAprotein encoded by the Mtb gene Rv3246c; and (e) an epitope of any of(a)-(d), said composition being substantially free of other M.tuberculosis proteins with which said early M. tuberculosis antigens arenatively admixed in a culture of M. tuberculosis.
 17. The antigeniccomposition of claim 16 wherein said other proteins are not early M.tuberculosis antigens.
 18. The antigenic composition of claim 16 or 17,further comprising one or more of: (a) an 88 kDa M. tuberculosis proteinhaving the an amino acid sequence SEQ ID NO:13: MTDRVSVGNL RIARVLYDFVNNEALPGTDI DPDSFWAGVD KVVADLTPQN QALLNARDEL QAQIDKWHRR RVIEPIDMDAYRQFLTEIGY LLPEPDDFTI TTSGVDAEIT TTAGPQLVVP VLNARFALNA ANARWGSLYDALYGTDVIPE TDGAEKGPTY NKVRGDKVIA YARKFLDDSV PLSSGSFGDA TGFTVQDGQLVVALPDKSTG LANPGQFAGY TGAAESPTSV LLINHGLHIE ILIDPESQVG TTDRAGVKDVILESAITTIM DFEDSVAAVD AADKVLGYRN WLGLNKGDLA AAVDKDGTAF LRVLNRDRNYTAPGGGQFTL PGRSLMFVRN VGHLMTNDAI VDTDGSEVFE GIMDALFTGL IAIHGLKASDVNGPLINSRT GSIYIVKPKM HGPAEVAFTC ELFSRVEDVL GLPQNTMKIG IMDEERRTTVNLKACIKAAA DRVVFINTGF LDRTGDEIHT SMEAGPMVRK GTMKSQPWIL AYEDHNVDAGLAAGFSGRAQ VGKGMWTMTE LMADMVETKI AQPRAGASTA WVPSPTAATL HALHVHQVDVAAVQQGLAGK RRATIEQLLT IPLAKELAWA PDEIREEVDN NCQSILGYVV RWVDQGVGCSKVPDIHDVAL MEDRATLRIS SQLLANWLRH GVITSADVRA SLERMAPLVD RQNAGDVAYRPMAPNFDDSI AFLAAQELIL SGAQQPNGYT EPILHRRRRE FKARAAEKPA PSDRAGDDAA R;

(b) a 27 kDa M. tuberculosis protein named MPT51 having the amino acidsequence SEQ ID NO:14 APYENLMVPS PSMGRDIPVA FLAGGPHAVY LLDAFNAGPDVSNWVTAGNA NTLAGKGIS VVAPAGGAYS MYTNWEQDGS KQWDTFLSAE LPDWLAANRGAAQGGYGAMA AAFHPDRFG FAGSMSGFLY PSNTTTNGAI AAGMQQFGGV DTNGMWGAPQLGRWKWHDPW HASLLAQNN TRVWVWSPTN PGASDPAAMI GQTAEAMGNS RMFYNQYRSVGGHNGHFDFP SGDNGWGSW APQLGAMSGD IVGAIR;

(c) a protein characterized as M. tuberculosis antigen 85C; or (d) aglycoprotein characterized as M. tuberculosis antigen MPT32.
 19. Theantigenic composition of any of claims 16-18 further comprising one ormore of the following M. tuberculosis antigenic proteins having anapproximate molecular weight as indicated: (i) a 28 kDa proteincorresponding to the spot identified as Ref. No. 77 in Table
 2. (ii) a29/30 kDa protein corresponding to the spot identified as Ref. No. 69 or59 in Table 2; (iii) a 31 kDa protein corresponding to the spotidentified as Ref. No. 103 in Table 2; (iv) a 35 kDa proteincorresponding to the spot identified as Ref. No. 66 in Table 2 andreacting with monoclonal antibody IT-23; (v) a 42 kDa proteincorresponding to the spot identified as Ref. No. 68 or 80 in Table 2;(vi) a 48 kDa protein corresponding to the spot identified as Ref. No.24 in Table 2; and (vii) a 104 kDa protein corresponding to the spotidentified as Ref. No. 111 in Table 2, which spots are obtained by2-dimensional electrophoretic separation of M. tuberculosislipoarabinomannan-free culture filtrate proteins as follows: (A)incubating 3 hours at 20° C. in 9M urea, 2% Nonidet P-40, 5%β-mercaptoethanol, and 5% ampholytes at pH 3-10; (B) isoelectricfocusing on 6% polyacrylamide isoelectric focusing tube gel of 1.5mm×6.5 cm, said gel containing 5% ampholytes in a 1:4 ratio of pH 3-10ampholytes to pH 4-6.5 ampholytes for 3 hours at 1 kV using 10 mM H₃PO₄as catholyte and 20 mM NaOH as anolyte, to obtain a focused gel; (C)subjecting the focused gel to SDS PAGE in the second dimension byplacement on a preparative SDS-polyacrylamide gel of 7.5×10 cm×1.5 mmcontaining a 6% stack over a 15% resolving gel and electrophoresing at20 mA per gel for 0.3 hours followed by 30 mA per gel for 1.8 hours.